Humanized Anti-CCR2 Antibodies and Methods of Use Therefor

ABSTRACT

The present invention relates to a humanized antibody or functional fragment thereof which binds to a mammalian (e.g., human) CC-chemokine receptor 2 (CCR2) or a portion of the receptor and blocks binding of a ligand to the receptor. The invention further relates to a method of inhibiting the interaction of a cell bearing mammalian CCR2 with a ligand thereof, and to use of the antibodies and fragments in therapeutic, prophylactic and diagnostic methods.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/497,625, filed Feb. 3, 2000, which is a continuation-in-part of U.S.application Ser. No. 09/359,193, filed Jul. 22, 1999, which is acontinuation-in-part of U.S. application Ser. No. 09/121,781, filed Jul.23, 1998, the entire teachings of all of which are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Over the past several years a growing family of leukocytechemoattractant/activating factors, termed chemokines, has beendescribed (Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648(1991); Schall and Bacon, Curr. Opin. Immunol., 6:865-873 (1994);Baggiolini, M., et al., Adv. Immunol., 55:97-179 (1994)). Members ofthis family are produced and secreted by many cell types in response toearly inflammatory mediators such as IL-1β or TNFα. The chemokinesuperfamily comprises two main branches: the α-chemokines (or CXCchemokines) and the β-chemokines (CC chemokines). The α-chemokine branchincludes proteins such as IL-8, neutrophil activating peptide-2 (NAP-2),melanoma growth stimulatory activity (MGSA/gro or GROα), and ENA-78,each of which have attracting and activating effects predominantly onneutrophils. The members of the β-chemokine branch affect other celltypes such as monocytes, lymphocytes, basophils, and eosinophils(Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991);Baggiolini, M., et al., Adv. Immunol., 55:97-179 (1994); Miller andKrangel, Crit. Rev. Immunol., 12:17-46 (1992); Jose, P. J., et al., J.Exp. Med., 179:881-118 (1994); Ponath, P. D., et al., J. Clin. Invest.,97:604-612 (1996)), and include proteins such as monocyte chemotacticproteins 1-4 (MCP-1, MCP-2, MCP-3, and MCP-4), RANTES, and macrophageinflammatory proteins (MIP-1α, MIP-1β). Recently a new class ofmembrane-bound chemokines designated CX3C chemokines has been identified(Bazan, J. F., et al., Nature 385:640-644 (1997)). Chemokines canmediate a range of pro-inflammatory effects on leukocytes, such astriggering of chemotaxis, degranulation, synthesis of lipid mediators,and integrin activation (Oppenheim, J. J. et al., Annu. Rev. Immunol.,9:617-648 (1991); Baggiolini, M., et al., Adv. Immunol., 55:97-179(1994); Miller, M. D. and Krangel, M. S., Crit. Rev. Immunol., 12:17-46(1992)). Lately, certain β-chemokines have been shown to suppress HIV-1infection of human T cell lines in vitro (Cocchi, F., et al., Science(Wash. DC), 270:1811-1815 (1995)).

Chemokines bind to 7 transmembrane spanning (7TMS) G protein-coupledreceptors (Murphy, P. M., Annu. Rev. Immunol., 12:593-633 (1994)). Someknown receptors for the CC or β chemokines include CCR1, which bindsMIP-1α and RANTES (Neote, K., et al., Cell, 72:415-425 (1993); Gao, J.L., J. Exp. Med., 177:1421-1427 (1993)); CCR2, which binds chemokinesincluding MCP-1, MCP-2, MCP-3 and MCP-4 (Charo, I. F., et al., Proc.Natl. Acad. Sci. USA, 91:2752-2756 (1994); Myers, S. J., et al., J.Biol. Chem., 270:5786-5792 (1995); Gong et al., J. Biol Chem272:11682-11685 (1997); Garcia-Zepeda et al., J. Immunol. 157:5613-5626(1996)); CCR3, which binds chemokines including eotaxin, RANTES andMCP-3 (Ponath, P. D., et al., J. Exp. Med., 183:2437-2448 (1996)); CCR4,which has been found to signal in response to MCP-1, MIP-1α, and RANTES(Power, C. A., et al., J. Biol. Chem., 270:19495-19500 (1995)); andCCR5, which has been shown to signal in response to MIP-1α, MIP-1β andRANTES (Boring, L., et al., J. Biol. Chem., 271 (13):7551-7558 (1996);Raport, C. J., J. Biol. Chem., 271:17161-17166 (1996); and Samson, M. etal., Biochemistry, 35:3362-3367 (1996)).

CCR2 is expressed on the surface of several leukocyte subsets, andappears to be expressed in two slightly different forms (CCR2a andCCR2b) due to alternative splicing of the mRNA encoding thecarboxy-terminal region (Charo et al., Proc. Natl. Acad. Sci. USA91:2752-2756 (1994)). MCP-1 acts upon monocytes, lymphocytes andbasophils, inducing chemotaxis, granule release, respiratory burst andhistamine and cytokine release. Studies have suggested that MCP-1 isimplicated in the pathology of diseases such as rheumatoid arthritis,atherosclerosis, granulomatous diseases and multiple sclerosis (Koch, J.Clin. Invest. 90:772-79 (1992); Hosaka et al., Clin. Exp. Immunol.97:451-457 (1994); Schwartz et al., Am. J. Cardiol. 71(6):9B-14B (1993);Schimmer et al., J. Immunol. 160:1466-1471 (1998); Flory et al., Lab.Invest. 69:396-404 (1993); Gong et al., J. Exp. Med. 186:131-137(1997)). Additionally, CCR2 can act as a co-receptor for HIV (Connor etal., J. Exp. Med. 185:621-628 (1997)). Thus, CCR2 receptor antagonistsmay represent a new class of important therapeutic agents.

SUMMARY OF THE INVENTION

The present invention relates to an antibody (immunoglobulin) orfunctional fragment thereof (e.g., an antigen-binding fragment) whichbinds to a mammalian CC-chemokine receptor 2 (also referred to as CCR2,CKR-2, MCP-1RA or MCP-1RB) or portion of the receptor (anti-CCR2). Inone embodiment, the antibody of the present invention or fragmentthereof has specificity for human or rhesus CCR2 or a portion thereof.In another embodiment, the antibody or fragment of the invention blocksbinding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptorand inhibits function associated with binding of the ligand to thereceptor (e.g., leukocyte trafficking). For example, as describedherein, antibodies and fragments thereof of the present invention whichbind human or rhesus CCR2 or a portion thereof, can block binding of achemokine (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptor and inhibitfunction associated with binding of the chemokine to the receptor. Inone embodiment, the antibody is monoclonal antibody (mAb) LS132.1D9(1D9) or an antibody which can compete with 1D9 for binding to humanCCR2 or a portion of human CCR2. Functional fragments of the foregoingantibodies are also envisioned.

In another embodiment, the antibody or functional fragment of thepresent invention binds human CCR2 or a portion thereof, and inhibitshuman immunodeficiency virus (HIV) binding to the receptor, therebyinhibiting function associated with binding of HIV to the receptor(e.g., HIV antigen release and infectivity). In one embodiment, theantibody is monoclonal antibody 1D9 or an antibody which can competewith 1D9 for binding to human CCR2 or a portion of human CCR2.

The present invention also relates to an antibody or functional fragmentthereof (e.g., an antigen-binding fragment) which binds to a mammalianCCR2 or portion of the receptor and provides increased fluorescentstaining intensity of CCR2 or compositions comprising CCR2 relative toother anti-CCR2 antibodies. In one embodiment, the antibody ismonoclonal antibody 1D9 or LS132.8G2 (8G2) or an antibody which cancompete with 1D9 or 8G2 for binding to human CCR2 or a portion of humanCCR2.

The present invention also relates to a humanized immunoglobulin orantigen-biding fragment thereof having binding specificity for CCR2,said immunoglobulin comprising an antigen binding region of nonhumanorigin (e.g., rodent) and at least a portion of an immunoglobulin ofhuman origin (e.g., a human framework region, a human constant region ofthe gamma type). In one embodiment, the humanized immunoglobulin orfragment thereof described herein can compete with 1D9 for binding toCCR2. In a preferred embodiment, the antigen binding region of thehumanized immunoglobulin is derived from monoclonal antibody 1D9 (e.g.,an immunoglobulin comprising the variable regions of the light and heavychains as shown in FIG. 7 (SEQ ID NO: 9) and FIG. 8 (SEQ ID NO: 10),respectively).

For example, the humanized immunoglobulin or antigen-binding fragmentthereof can comprise an antigen binding region comprising at least onecomplementarity determining region (CDR) of nonhuman origin, and aframework region (FR) derived from a human framework region. In oneaspect, the humanized immunoglobulin having binding specificity for CCR2comprises a light chain comprising at least one CDR derived from anantibody of nonhuman origin which binds CCR2 and a FR derived from alight chain of human origin (e.g., from HF-21/28), and a heavy chaincomprising a CDR derived from an antibody of nonhuman origin which bindsCCR2 and a FR derived from a heavy chain of human origin (e.g., from4B4′CL). In another aspect, the light chain comprises three CDRs derivedfrom the light chain of the 1D9 antibody, and the heavy chain comprisesthree CDRs derived from the heavy chain of the 1D9 antibody.

The present invention also relates to humanized immunoglobulin lightchains and antigen-binding fragments thereof (e.g., comprising CDR1,CDR2 and CDR3 of the light chain of the 1D9 antibody, and a human lightchain FR), and to humanized immunoglobulin heavy chains andantigen-binding fragments thereof (e.g., comprising CDR1, CDR2 and CDR3of the heavy chain of the 1D9 antibody, and a human heavy chain FR). Ina preferred embodiment, the invention relates to humanized heavy andlight chains described herein (e.g., a humanized light chain comprisingthe variable region of the light chain shown in FIG. 7 (SEQ ID NO: 9), ahumanized heavy chain comprising the variable region of the heavy chainshown in FIG. 8 (SEQ ID NO: 10). Also encompassed are humanizedimmunoglobulins comprising one or more humanized light and/or heavychains.

The invention further relates to isolated nucleic acid moleculescomprising a nucleic acid sequence which encodes a humanizedimmunoglobulin of the present invention (e.g., a single chain antibody),as well as to isolated nucleic acid molecules comprising a sequencewhich encodes a humanized immunoglobulin light chain (e.g., comprisingnucleotides 52-390 of SEQ ID NO: 95) or heavy chain (e.g., comprisingnucleotides 58-411 of SEQ ID NO: 96) of the present invention. Forexample, the present invention provides a gene (e.g., a fused gene)encoding a humanized immunoglobulin light or heavy chain comprising afirst nucleic acid sequence encoding an antigen binding region derivedfrom murine 1D9 monoclonal antibody; and a second nucleic acid sequenceencoding at least a portion of a constant region of an immunoglobulin ofhuman origin.

The present invention further relates to a construct comprising anucleic acid molecule encoding a humanized immunoglobulin having bindingspecificity for CCR2 or a chain of such an immunoglobulin. For example,an expression vector comprising a gene (e.g., a fused gene) encoding ahumanized immunoglobulin light chain, comprising a nucleotide sequenceencoding a CDR derived from a light chain of a nonhuman antibody havingbinding specificity for CCR2, and a framework region derived from alight chain of human origin, is provided. An expression vectorcomprising a gene encoding a humanized immunoglobulin heavy chain,comprising a nucleotide sequence encoding a CDR derived from a heavychain of a nonhuman antibody having binding specificity for CCR2, and aframework region derived from a heavy chain of human origin is anotherexample of such a construct.

The present invention also relates to a host cell comprising a nucleicacid molecule of the present invention, including one or more constructscomprising a nucleic acid molecule of the present invention. In oneembodiment, the invention relates to a host cell comprising a firstrecombinant nucleic acid encoding a humanized immunoglobulin lightchain, and a second recombinant nucleic acid encoding a humanizedimmunoglobulin heavy chain, said first nucleic acid comprising anucleotide sequence encoding a CDR derived from the light chain ofmurine 1D9 antibody and a framework region derived from a light chain ofhuman origin; and said second nucleic acid comprising a nucleotidesequence encoding a CDR derived from the heavy chain of murine 1D9antibody and a framework region derived from a heavy chain of humanorigin.

The present invention also provides a method of preparing a humanizedimmunoglobulin comprising maintaining a host cell of the presentinvention under conditions appropriate for expression of a humanizedimmunoglobulin, whereby a humanized immunoglobulin chain(s) is expressedand a humanized immunoglobulin is produced. The method can furthercomprise the step of isolating the humanized immunoglobulin.

The humanized immunoglobulins of the present invention can be lessimmunogenic than their murine or other nonhuman counterparts. Thus, thehumanized immunoglobulins described herein can be used as therapeuticagents in humans, for example to control lymphocyte homing to mucosallymphoid tissue, thereby, reducing inflammatory responses.

The invention further relates to a humanized immunoglobulin of thepresent invention for use in diagnosis or therapy (includingprophylaxis). In one embodiment, the invention relates to a humanizedimmunoglobulin of the present invention for use in the treatment ofdiseases associated with leukocyte infiltration of tissues, for example,in the treatment of inflammatory diseases, autoimmune diseases, graftrejection, HIV infection and monocyte-mediated disorders such asatherosclerosis.

In another aspect, the invention relates to use of a humanizedimmunoglobulin of the present invention for the manufacture of amedicament for the treatment of diseases associated with leukocyteinfiltration of tissues, for example, in the treatment of inflammatorydiseases, autoimmune diseases, monocyte-mediated disorders such asatherosclerosis, graft rejection, or HIV infection.

The present invention further relates to a method of inhibiting theinteraction of a cell bearing mammalian (e.g., human, non-human primateor murine) CCR2 with a ligand thereof, comprising contacting the cellwith an effective amount of an antibody or functional fragment thereofwhich binds to a mammalian CCR2 or a portion of CCR2. Suitable cellsinclude granulocytes, leukocytes, such as monocytes, macrophages,basophils and eosinophils, mast cells, and lymphocytes including T cells(e.g., CD8+ cells, CD4+ cells, CD25+ cells, CD45RO+ cells), and othercells expressing CCR2 such as a recombinant cell expressing CCR2 (e.g.,transfected cells). In a particular embodiment, the antibody is 1D9 oran antibody which can compete with 1D9 for binding to human CCR2 or aportion of human CCR2.

Another embodiment of the invention relates to a method of inhibitingthe interaction of a cell bearing mammalian CCR2 with a chemokine,comprising contacting said cell with an effective amount of an antibodyor functional fragment thereof which binds to CCR2 or a portion of saidreceptor. In one embodiment of the method, the antibody or functionalfragment thereof is any one or more of 1D9, an antigen-binding fragmentof 1D9 or an antibody or fragment thereof having an epitopic specificitywhich is the same as or similar to that of 1D9. Furthermore, theinvention relates to a method of inhibiting a function associated withbinding of a chemokine to CCR2, comprising administering an effectiveamount of an antibody or functional fragment thereof which binds to amammalian CCR2 protein or a portion of said receptor. In one aspect ofthe method, the antibody or functional fragment thereof is any one ormore of 1D9, an antigen-binding fragment of 1D9 or an antibody orfragment thereof having an epitopic specificity which is the same as orsimilar to that of 1D9.

Another aspect of the invention is a method of identifying expression ofa mammalian CCR2 or portion of the receptor by a cell. According to themethod, a composition comprising a cell or fraction thereof (e.g., amembrane fraction) is contacted with an antibody or functional fragmentthereof (e.g., 1D9 or 8G2) which binds to a mammalian CCR2 protein orportion of the receptor under conditions appropriate for binding of theantibody thereto, and the formation of a complex between said antibodyor fragment and said protein or portion thereof is detected. Detectionof the complex, directly or indirectly, indicates the presence of thereceptor on the cell. The present invention also relates to a kit foruse in detecting the presence of CCR2 or a portion thereof in abiological sample, comprising an antibody or functional fragment thereofwhich binds to a mammalian CC-chemokine receptor 2 or a portion of saidreceptor, and one or more ancillary reagents suitable for detecting thepresence of a complex between said antibody or fragment and said proteinor portion thereof.

Also encompassed by the present invention are methods of identifyingadditional ligands or other substances which bind a mammalian CCR2protein, including inhibitors and/or promoters of mammalian CCR2function. For example, agents having the same or a similar bindingspecificity as that of an antibody of the present invention orfunctional fragment thereof can be identified by a competition assaywith said antibody or fragment. Thus, the present invention alsoencompasses methods of identifying ligands or other substances whichbind the CCR2 receptor, including inhibitors (e.g., antagonists) orpromoters (e.g., agonists) of receptor function. In one embodiment,cells which naturally express CCR2 receptor protein or suitable hostcells which have been engineered to express a CCR2 receptor or variantencoded by a nucleic acid introduced into said cells are used in anassay to identify and assess the efficacy of ligands, inhibitors orpromoters of receptor function. Such cells are also useful in assessingthe function of the expressed receptor protein or polypeptide.

Thus, the invention also relates to a method of detecting or identifyingan agent which binds a mammalian CCR2 or ligand binding variant thereof,comprising combining an agent to be tested, an antibody orantigen-binding fragment of the present invention (e.g., monoclonalantibody 1D9, an antibody having an epitopic specificity which is thesame as or similar to that of 1D9, antigen-binding fragments of 1D9,monoclonal antibody 8G2, an antibody having an epitopic specificitywhich is the same as or similar to that of 8G2, and antigen-bindingfragments of 8G2) and a composition comprising a mammalian CCR2 proteinor a ligand binding variant thereof. The foregoing components can becombined under conditions suitable for binding of the antibody orantigen-binding fragment to mammalian CCR2 protein or a ligand bindingvariant thereof, and binding of the antibody or fragment to themammalian CCR2 protein or ligand binding variant is detected ormeasured, either directly or indirectly, according to methods describedherein or other suitable methods. A decrease in the amount of complexformed relative to a suitable control (e.g., in the absence of the agentto be tested) is indicative that the agent binds said receptor orvariant. The composition comprising a mammalian CCR2 protein or a ligandbinding variant thereof can be a membrane fraction of a cell bearingrecombinant CCR2 protein or ligand binding variant thereof. The antibodyor fragment thereof can be labeled with a label such as a radioisotope,spin label, antigen label, enzyme label, fluorescent group andchemiluminescent group. These and similar assays can be used to detectagents, including ligands (e.g., chemokines which interact with CCR2) orother substances, including inhibitors or promoters of receptorfunction, which can bind CCR2 and compete with the antibodies describedherein for binding to the receptor.

According to the present invention, ligands, inhibitors or promoters ofreceptor function can be identified in a suitable assay, and furtherassessed for therapeutic effect. Inhibitors of receptor function can beused to inhibit (reduce or prevent) receptor activity, and ligandsand/or promoters can be used to induce (trigger or enhance) normalreceptor function where indicated. The present invention also provides amethod of treating inflammatory diseases, autoimmune diseases,atherosclerosis, and graft rejection, or HIV infection, comprisingadministering an inhibitor of receptor function (e.g., chemokine bindingor HIV binding) to an individual (e.g., a mammal, such as a human). Thepresent invention further provides a method of stimulating receptorfunction by administering a novel ligand or promoter to an individual,providing a new approach to selective stimulation of leukocyte function,which is useful, for example, in the treatment of infectious diseasesand cancer.

Another aspect of the invention relates to a method of inhibiting HIVinfection of a cell which expresses a mammalian CCR2 or portion thereof,comprising contacting the cell with an effective amount of an antibodyor functional fragment thereof which binds to a mammalian CCR2 orportion of the receptor and inhibits HIV binding and infection. In aparticular embodiment of the invention, the antibody or functionalfragment thereof is any of 1D9, an antibody having an epitopicspecificity which is the same as or similar to that of 1D9, an antibodywhich can compete with 1D9 for binding to human CCR2, andantigen-binding fragments thereof.

Also encompassed by the present invention is a method of inhibiting(e.g., treating) HIV in a patient, comprising administering to thepatient an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 or a portion of said receptorand inhibits HIV binding to the CCR2 receptor. The anti-CCR2 antibody orfragment can be administered alone or in combination with one or moreadditional therapeutic agents, e.g., one or more antibodies which bind aco-receptor for HIV infection and inhibit binding to said co-receptor,such as an anti-CCR3, anti-CCR5, and/or anti-CXCR4 antibody.

Another aspect of the invention also relates to a method of preventingor inhibiting HIV infection in an individual, comprising administeringto the individual an effective amount of an antibody or functionalfragment thereof which binds to CCR2 and inhibits HIV binding to CCR2.According to the method, preventing HIV infection includes treatment inorder to prevent (reduce or eliminate) infection of new cells in aninfected individual or in order to prevent infection in an individualwho may be, may have been or has been exposed to HIV. For example,individuals such as an HIV infected individual, a fetus of an HIVinfected female, or a health care worker can be treated according to themethod of the present invention.

The present invention also encompasses a method of inhibiting leukocytetrafficking in a patient, comprising administering to the patient aneffective amount of an antibody or functional fragment thereof whichbinds to a mammalian CCR2 or portion of said receptor and inhibitsfunction associated with binding of a ligand to the receptor.

The present invention also relates to a method of inhibiting or treatingCCR2-mediated disorders, such as inflammatory disorders, comprisingadministering to a patient an effective amount of an antibody orfunctional fragment thereof which binds to a mammalian CCR2 or portionof said receptor and inhibits CCR2-mediated function. For example, theinvention relates to a method of inhibiting or treating stenosis orrestenosis of the vasculature comprising administering to a patient aneffective amount of an antibody or functional fragment thereof whichbinds to a mammalian CCR2 or portion of said receptor and inhibitsCCR2-mediated function.

The present invention further relates to an antibody or fragment thereofas described herein (e.g., monoclonal antibody 1D9 or an antigen-bindingfragment thereof) for use in therapy (including prophylaxis) ordiagnosis, and to the use of such an antibody or fragment for themanufacture of a medicament for the treatment of a CCR2-mediateddisorder, or other disease or inflammatory condition as describedherein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1O are fluorescence activated cell scanning (FACS) histogramprofiles illustrating that mAbs 1D9 and 8G2 stain CCR2 transfectants butnot CCR5 or CCR1 transfectants. L1/2 (also referred to herein as L1.2)murine pre-B lymphoma host cells were transfected with CCR2, CCR5 andCCR1 as indicated, and stained with antibodies with different receptorspecificities: Staining was analyzed by flow cytometry.

FIGS. 2A-2L are FACS dot plots showing expression of CCR2 on mostmonocytes, a subpopulation of lymphocytes and a small subset ofgranulocytes. Whole blood cells were stained with one of three anti-CCR2mAbs (5A11, generated using a peptide consisting of the first 32 aminoacids of the CCR2 amino-terminus as an immunogen, and 1D9 and 8G2generated as described herein using CCR2b L1/2 cell transfectants as theimmunogen). Staining was analyzed by flow cytometry, and the lymphocyte,granulocyte and monocyte populations were gated using the forward andside light scatter. The X-axis represents forward light scatter (ameasure of cell size), and the Y-axis fluorescence intensity of stainingfor CCR2. The level of negative control staining is indicated by a line.

FIGS. 3A-3I are FACS dot plots showing that mAb 1D9 stains an IgEpositive population in peripheral blood (basophils) using two-colorstaining for IgE and CCR2. Whole blood cells were first stained witheither a negative control antibody (anti-Flag), anti-CCR2 antibody 1D9,or an anti-CXCR1 antibody, as indicated, and detected by ananti-mouse-FITC conjugate. A second staining was done using either PBSor a biotinylated antibody specific for IgE or CD16, as indicated, anddetected with a streptavidin-phycoerythrin. Staining was analyzed byflow cytometry.

FIG. 4 illustrates that mAb 1D9 inhibits [¹²⁵I]MCP-1 binding to THP-1cell membranes. 3.0 μg of THP-1 membrane protein was incubated with 0.1nM [¹²⁵I]MCP-1 in the presence of various concentrations of 1D9 or theisotype-matched anti-CXCR3 antibody 1C6. The amount of bound tracer wasdetermined by separation of free from bound by filtration andscintillation counting. The data was analyzed to determine the IC₅₀value by non-linear regression using a 4-parameter logistic equationwith KaleidaGraph software.

FIG. 5 illustrates that mAb 1D9 inhibits [¹²⁵I]MCP-1 binding to freshhuman PBMC. Freshly isolated peripheral blood mononuclear cells(500,000) were incubated with 0.1 nM [¹²⁵I]MCP-1 in the presence ofvarious concentrations of 1D9 or the isotype-matched anti-CXCR3 antibody1C6. The amount of bound tracer was determined by separation of freefrom bound by filtration and scintillation counting. The data wasanalyzed to determine the IC₅₀ value as for FIG. 4.

FIGS. 6A and 6B are graphs demonstrating that mAb 1D9 inhibitsMCP-1-induced chemotaxis, but not RANTES-induced chemotaxis, of freshPBMC. FIG. 6A shows the results of chemotaxis assays of PBMC to 10 nMMCP-1 with no antibody, or 0.1 or 10 μg/ml of 1D9 or nonspecific murineIgG2a. The spontaneous nonspecific migration is also indicated. FIG. 6Bshows the results of chemotaxis assays of PBMC to 10 nM RANTES with noantibody, 10 μg/ml 1D9 or 10 μg/ml nonspecific murine IgG2a. Thespontaneous nonspecific migration in the absence of RANTES is alsoindicated.

FIG. 7 shows the amino acid sequence (SEQ ID NO: 9) of the kappa lightchain variable region of the murine 1D9 antibody. The CDRs arehighlighted in bold.

FIG. 8 shows the amino acid sequence (SEQ ID NO: 10) of the heavy chainvariable region of the murine 1D9 antibody. The CDRs are highlighted inbold.

FIG. 9 illustrates the canonical classes of CDRs in the murine 1D9 V_(K)region. “Chothia Canonical Classes” indicates where the canonicalclasses as defined by Chothia and his colleagues (Chothia and Lesk, J.Mol. Biol. 197:901 (1987); Chothia et al., Nature 34:877 (1989);Tramontano et al., J. Mol. Biol. 215:175 (1990); and Chothia et al., J.Mol. Biol. 227:799 (1992)) were used, while “Martin Canonical Classes”signifies where the canonical classes defined by Martin and Thornton(Martin and Thornton, J: Mol. Biol. 263:800 (1996)) were used. FRresidues are highlighted in bold.

FIG. 10 illustrates the canonical classes of CDRs in the murine 1D9V_(H) region. “Chothia Canonical Classes” indicates where the canonicalclasses as defined by Chothia and his colleagues (Chothia and Lesk, J.Mol. Biol. 197:901 (1987); Chothia et al., Nature 34:877 (1989);Tramontano et al., J. Mol. Biol. 215:175 (1990); and Chothia et al., J.Mol. Biol. 227:799 (1992)) were used, while “Martin Canonical Classes”signifies where the canonical classes defined by Martin and Thornton(Martin and Thornton, J. Mol. Biol. 263:800 (1996)) were used. FRresidues are highlighted in bold.

FIG. 11 shows the amino acid sequences of various versions of thehumanised 1D9 V_(K) region (SEQ ID NOS: 12-15, respectively). Where the1D9 V_(K) region residues (SEQ ID NO: 9) and the human HF-21/28 V_(K)region (SEQ ID NO: 11) sequences match a dot [.] is shown. Where noamino acid is present at a specific residue position a dash [-] isshown. Where an amino acid in the HF-21/28 FRs is changed in thehumanised 1D9 V_(K) region, it is highlighted in bold. The CDRs aredescribed by the use of nomenclature [==L1==]. The numbering used isaccording to Kabat et al., Sequences of proteins of immunologicalinterest, Fifth edition, U.S. Department of Health and Human Services,U.S. Government Printing Office (1991).

FIG. 12 shows the amino acid sequences of various versions of thehumanised 1D9 V_(H) region (SEQ ID NOS: 17-20, respectively). Where the1D9 V_(H) region residues (SEQ ID NO: 10) and the human 4B4′CL V_(H)region sequences (SEQ ID NO: 16) match a dot [.] is shown. Where noamino acid is present at a specific residue position a dash [-] isshown. Where an amino acid in the 4B4′CL is changed in the humanised 1D9V_(H) region, it is highlighted in bold. The CDRs are described by theuse of nomenclature [==H==], while [-----] denotes part of the H1structure loop. The numbering used is according to Kabat et al.,Sequences of proteins of immunological interest, Fifth edition, U.S.Department of Health and Human Services, U.S. Government Printing Office(1991).

FIG. 13 shows a comparison of a portion of the murine 1D9 V_(K) region(SEQ ID NO: 21) with mouse germline V_(K) gene sequences (SEQ ID NOS:22-33, respectively). “Identical residues” represents the number ofidentical residues in a mouse germline V_(K) region to the murine 1D9V_(K) region. Where the 1D9 V_(K) region sequence and the mouse germlineV_(K) region sequences match a dot [.] is shown. Where no amino acid ispresent at a specific residue position a dash [-] is shown.

FIG. 14 shows a comparison of a portion of the murine 1D9 V_(H) region(SEQ ID NO: 34) with mouse germline V_(H) gene sequences (SEQ ID NOS:35-53, respectively). “Identical residues” represents the number ofidentical residues in a mouse germline V_(H) region to the murine 1D9V_(H) region. Where the 1D9 V_(H) region sequence and the mouse germlineV_(H) region sequences match a dot [.] is shown. Where no amino acid ispresent at a specific residue position a dash [-] is shown.

FIG. 15 shows a comparison of the murine 1D9 V_(K) region (SEQ ID NO: 9)with the most homologous seventeen human V_(K) amino acid sequences (SEQID NOS: 54-70, respectively). “ID” represents the percentage identity ofthe human V_(K) sequences to the murine 1D9 V_(K) region. Where the 1D9V_(K) region residues and the human V_(K) region sequences match a dot[.] is shown. Where no amino acid is present at a specific residueposition a dash [-] is shown. “S” indicates amino acid positions on thesurface of the F_(V) domain. “C” indicates residues located within thecore of the F_(V) domain. Residues within 5A of a CDR are defined usingcapital letters, while those located further away are described with alower case letter. The CDRs themselves are described by the use of thenomenclature ==L1==. “v” denotes the Vernier residues (Foote and Winter,J. Mol. Biol. 224:487 (1992)) located in the FRs. Those residues in thehuman V_(K) region sequences which are underlined differ from theirclosest human V_(K) germline gene. The numbering used is as according toKabat et al., Sequences of proteins of immunological interest, Fifthedition, U.S. Department of Health and Human Services, U.S. GovernmentPrinting Office (1991).

FIG. 16 shows a comparison of the murine 1D9 V_(K) region with the mosthomologous seventeen human V_(K) amino acid sequences. “ID” indicatesthe percentage identity of the human V_(K) sequence to the murine 1D9V_(K) region. “Surface” indicates the number of identical residues onthe surface. “Core” indicates the number of identical residues withinthe core of the F_(V) domain. “CDR” indicates the number of identicalresidues within the CDRs. “FR” indicates the number of identicalresidues within the FRs. “FR Surface” indicates the number of identicalresidues which are surface exposed. “FR Core” indicates the number ofidentical residues which are located within the core of the F_(V)domain. “FR Near CDR” represents the number of identical residuesamongst the FR amino acids within 5A of a CDR. “Vernier” indicates thenumber of identical residues amongst the 14 Vernier amino acids (Footeand Winter, J. Mol. Biol. 224:487 (1992)). “V_(K)” indicates the numberof identical residues within the V_(K) gene. “J Chain” indicates thenumber of identical residues within the J chain gene. “L1 Len” to “L3Len” defines the number of residues in each CDR, while “L1 Class” to “L3Class” describes the canonical class of the CDR according to Martin andThornton (Martin and Thornton, J. Mol. Biol. 263:800 (1996)).

FIGS. 17A-17B show a comparison of the murine 1D9 V_(H) region (SEQ IDNO: 10) with the most homologous 24 human V_(H) amino acid sequences(SEQ ID NOS: 71-94, respectively). “ID” represents the percentageidentity of the human V_(H) sequences to the murine 1D9 V_(H) region.Where the 1D9 V_(H) region residues and the human V_(H) region sequencesmatch a dot [.] is shown. Where no amino acid is present at a specificresidue position a dash [-] is shown. “S” indicates amino acid positionson the surface of the F_(V) domain. “C” indicates residues locatedwithin the core of the F_(V) domain. Residues within 5A of a CDR aredefined using capital letters, while those located farther away aredescribed with a lower case letter. The CDRs themselves are described bythe use of the nomenclature ==H1==. “v” denotes the Vernier residues(Foote and Winter, J. Mol. Biol. 224:487 (1992)) located in the FRs.Those residues in the human V_(H) region sequences which are underlineddiffer from their closest human V_(H) germline gene. The numbering usedis as according to Kabat et al., Sequences of proteins of immunologicalinterest, Fifth edition, U.S. Department of Health and Human Services,U.S. Government Printing Office (1991).

FIGS. 18A-18B show a comparison of the murine 1D9 V_(H) region with themost homologous 24 human V_(H) amino acid sequences. “ID” indicatespercentage identity of the human V_(H) sequence to the murine 1D9 V_(H)region. “All” indicates the number of identical residues in the whole ofthe human V_(H) region when compared to the whole of the murine 1D9V_(H) region. “Surface” indicates the number of identical residues onthe surface. “Core” indicates the number of identical residues withinthe core of the F_(V) domain. “CDR” indicates the number of identicalresidues within the CDRs. “FR” indicates the number of identicalresidues within the Frs. “FR Surface” indicates the number of identicalresidues which are surface exposed. “FR Core” indicates the number ofidentical residues which are located within the core of the F_(V)domain. “FR Near CDR” represents the number of identical residuesamongst the FR amino acids within 5A of a CDR. “Vernier” indicates thenumber of identical residues amongst the 14 Vernier amino acids (Footeand Winter, J. Mol. Biol. 224:487 (1992)). “V_(H)” indicates the numberof identical residues within the V_(H) gene. “J Chain” indicates thenumber of identical residues within the J chain gene. “H1 Size” to “H3Size” define the number of residues in each CDR, while “H1 Class” and“H2 Class” describe the canonical class of the CDR according to Martinand Thornton, (J. Mol. Biol. 263:800 (1996)).

FIGS. 19A-19C show the alignment of amino acid sequences leading to thedesign of the first (1D9RK_(A) and second (1D9RK_(B)) humanised versionsof the 1D9 antibody kappa light chain variable region. Amino acidsidentical to the mouse 1D9 at a particular residue position in column 7are not shown; “-” indicates no amino acid is located at this residueposition. Boldface type indicates positions in FRs and CDRs where thehuman amino acid residue was replaced by the corresponding mouseresidue. “Δ” indicates the numbering of changes in the human FRs of1D9RK_(A). “Mouse 1D9 V_(K)” indicates the amino acid sequence of theV_(K) region from the murine 1D9 kappa light chain variable region.“Mouse κ-II” indicates the consensus sequence of mouse V_(K) regionsfrom Kabat subgroup κ-II. “Human κ-II” indicates the consensus sequenceof human V_(K) regions from Kabat subgroup κ-II. “HF-21/28” indicatesthe amino acid sequence of the light chain variable region from thehuman HF-21/28 antibody (Chastagner et al., Gene 101(2):305-6 (1991)).The number in parenthesis (005056) is the Kabat database ID number.“Surface or Core” indicates the position of the amino acid in relationto the rest of the residues in both chains of the antibody variableregions. Residues within 5A of a CDR are defined using capital letters.“1D9RK_(A)” indicates the amino acid sequence of the first version ofthe humanised 1D9 V_(K) region. “1D9RK_(B)” indicates the amino acidsequence of the second version of the humanised 1D9 V_(K) region.

FIGS. 20A-20C show the alignment of amino acid sequences leading to thedesign of the first (1D9RH_(A)) and second (1D9RH_(B)) humanised humanversions of the 1D9 antibody kappa heavy chain variable region. Aminoacids identical to the mouse 1D9 at a particular residue position incolumn 7 are not shown. “-” indicates that no amino acid is located atthis residue position. Boldface type indicates positions in the FRs andCDRs where the human amino acid residue was replaced by thecorresponding mouse residue. “Δ” indicates the numbering of changes inthe human FRs of 1D9RH_(A). “Mouse 1D9 V_(H)” indicates the amino acidsequence of the V_(H) region from the murine 1D9 heavy chain variableregion. “Mouse IIIc” indicates the consensus sequence of mouse V_(H)regions from Kabat subgroup IIIc. “Human III” indicates the consensussequence of human V_(H) regions from Kabat subgroup III. “4B4′CL”indicates the amino acid sequence of the heavy chain variable regionfrom the human 4B4′CL antibody (Sanz et al., Journal of Immunology142:883 (1989)). The number in parenthesis (000490) is the Kabatdatabase ID number. “Surface or Core” indicates the position of theamino acid in relation to the rest of the residues in both chains of theantibody variable regions. Residues within 5A of a CDR are defined usingcapital letters. “1D9RH_(A)” indicates the amino acid sequence of thefirst version of the humanised 1D9 V_(H) region. “1D9RH_(B)” indicatesthe amino acid sequence of the second version of the humanised 1D9 V_(H)region.

FIG. 21 shows the nucleotide sequence, complement and encoded amino acidsequence of the murine antibody 1D9 heavy chain variable region. Theleader sequence and a portion of the constant region are also shown. Theillustrated nucleotide sequence is SEQ ID NO: 96, the complementarysequence is SEQ ID NO: 99, and the amino acid sequence is SEQ ID NO:100.

FIG. 22 shows the nucleotide sequence, complement and encoded amino acidsequence of the murine antibody 1D9 kappa light chain variable region.The leader sequence and a portion of the constant region are also shown.The illustrated nucleotide sequence is SEQ ID NO: 95, the complementarysequence is SEQ ID NO: 101, and the amino acid sequence is SEQ ID NO:102.

FIG. 23 shows the nucleotide sequence of the humanized heavy chain1D9RH_(A). The indicated enzyme sites were added for cloning into thevector pLKTOK38. The vector also has human leader and constant regions.The illustrated nucleotide sequence is SEQ ID NO: 97, the complementarysequence is SEQ ID NO: 103, and the amino acid sequence is SEQ ID NO:104.

FIG. 24 shows the nucleotide sequence of the humanized light chain1D9RK_(A). The indicated enzyme sites were added for cloning into thevector pLKTOK38. The vector also has human leader and constant regions.The bracketed Y indicates a residue which changes to aspartate when putinto the vector pLKTOK38. The illustrated nucleotide sequence is SEQ IDNO: 98, the complementary sequence is SEQ ID NO: 105, and the amino acidsequence is SEQ ID NO: 106.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an antibody (anti-CCR2) or functionalfragment thereof which binds mammalian CC-chemokine receptor 2 (CCR2,CKR-2, MCP-1RA or MCP-1RB) or a portion of CCR2. In one embodiment, theantibody has specificity for human or rhesus CCR2 or portion thereof. Inone embodiment, the antibodies (immunoglobulins) are raised against anisolated and/or recombinant mammalian CCR2 or portion thereof (e.g.,peptide) or against a host cell which expresses mammalian CCR2. In apreferred embodiment, the antibodies specifically bind human CCR2receptor(s) (e.g., CCR2a and/or CCR2b) or a portion thereof, and in aparticularly preferred embodiment the antibodies have specificity for anaturally occurring or endogenous human CCR2. As used herein,“CC-chemokine receptor 2” (“CCR2”) refers to CC-chemokine receptor 2aand/or CC-chemokine receptor 2b. Antibodies or functional fragmentsthereof which can inhibit one or more functions characteristic of amammalian CCR2, such as a binding activity (e.g., ligand, inhibitorand/or promoter binding), a signaling activity (e.g., activation of amammalian G protein, induction of a rapid and transient increase in theconcentration of cytosolic free calcium [Ca²⁺]i), and/or stimulation ofa cellular response (e.g., stimulation of chemotaxis, exocytosis orinflammatory mediator release by leukocytes, integrin activation) arealso encompassed by the present invention, such as an antibody which caninhibit binding of a ligand (i.e., one or more ligands) to CCR2 and/orone or more functions mediated by CCR2 in response to a ligand. Forexample, in one aspect, the antibodies or functional fragments thereofcan inhibit (reduce or prevent) the interaction of receptor with anatural ligand, such as MCP-1, MCP-2, MCP-3 and/or MCP-4. In anotheraspect, an antibody or functional fragment thereof that binds to CCR2can inhibit binding of MCP-1, MCP-2, MCP-3 and/or MCP-4 and/or HIV tomammalian CCR2 (e.g., human CCR2, non-human primate CCR2, murine CCR2).The antibodies or functional fragments thereof of the present inventioncan inhibit functions mediated by human CCR2, including leukocytetrafficking, HIV entry into a cell, T cell activation, inflammatorymediator release and/or leukocyte degranulation. Preferably, theantibodies or fragments can bind CCR2 with an affinity of at least about0.1×10⁻⁹ M, preferably at least about 1×10⁻⁹ M, and more preferably atleast about 3×10⁻⁹ M. In a particular embodiment, antibodies orfunctional fragments thereof demonstrate inhibition of chemokine-induced(e.g., MCP-1-induced) chemotaxis of cells (e.g., PBMC) at less thanabout 150 μg/ml, preferably less than about 100 μg/ml, more preferablyless than about 50 μg/ml, and even more preferably less than about 20μg/ml.

In a further embodiment of the invention, the antibodies or functionalfragments thereof of the invention can inhibit binding of a CCR2 ligand(e.g., a chemokine) to CCR2 with an IC₅₀ of less than about 1.0 μg/ml,preferably less than about 0.05 μg/ml, and more preferably less thanabout 0.005 μg/ml.

Murine monoclonal antibodies specific for CCR2, designated 1D9 and 8G2,were produced as described herein. In a preferred embodiment, theantibodies of the present invention bind human CCR2, and have anepitopic specificity which is the same as or similar to that of murine1D9 or 8G2 antibody described herein. Antibodies with an epitopicspecificity which is the same as or similar to that of murine 1D9monoclonal antibody can be identified by their ability to compete withmurine 1D9 monoclonal antibody for binding to human CCR2 (e.g., to cellsbearing human CCR2, such as transfectants bearing CCR2, CD8+ cells, CD4+cells, CDR45RO+ cells, CD25+ cells, monocytes, dendritic cells,macrophages and basophils). Similarly, antibodies with an epitopicspecificity which is the same as or similar to that of murine 8G2monoclonal antibody can be identified by their ability to compete withmurine 8G2 monoclonal antibody for binding to human CCR2. Using receptorchimeras (Rucker et al., Cell 87:437-446 (1996)), the binding site ofmAbs 1D9 and 8G2 has been mapped to the amino-terminal domain of humanCC-chemokine receptor 2, specifically to an epitope comprising fromabout amino acid 1 to about amino acid 30 of the protein. Using these orother suitable techniques, antibodies having an epitopic specificitywhich is the same as or similar to that of an antibody of the presentinvention can be identified. mAbs 1D9 and 8G2 have epitopic specificityfor the amino-terminal domain of the CCR2 receptor, e.g., from aboutamino acid number 1 to about amino acid number 30 of the receptorprotein. Thus, the invention pertains to an antibody or functionalportion thereof which binds to the amino-terminal domain or portionthereof of mammalian CC-chemokine receptor 2, and particularly to anepitope comprising from about amino acid 1 to about amino acid 30 ofmammalian CC-chemokine receptor 2.

The invention also relates to a bispecific antibody, or functionalfragment thereof (e.g., F(ab′)₂), which has the same or similar epitopicspecificity as at least two of the antibodies described herein (see,e.g., U.S. Pat. No. 5,141,736 (Iwasa et al.), U.S. Pat. Nos. 4,444,878,5,292,668, 5,523,210 (all to Paulus et al.) and U.S. Pat. No. 5,496,549(Yamazaki et al.). For example, a bispecific antibody of the presentinvention can have the same or similar epitopic specificity as mAb 1D9and 8G2, e.g., binds the amino terminal domain, or portion thereof, ofmammalian CCR2 protein.

Hybridoma cell lines producing antibodies according to the presentinvention were deposited on Jul. 17, 1998, on behalf of LeukoSite, Inc.,215 First Street, Cambridge, Mass. 02142, U.S.A., at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20110,U.S.A., under Accession Nos. HB-12549 (1D9) and HB-12550 (8G2). Thepresent invention also pertains to the hybridoma cell lines depositedunder ATCC Accession No. HB-12549 and ATCC Accession No. HB-12550, aswell as to the monoclonal antibodies produced by the hybridoma celllines deposited under ATCC Accession Nos. HB-12549 and HB-12550.

The antibodies of the present invention can be polyclonal or monoclonal,and the term “antibody” is intended to encompass both polyclonal andmonoclonal antibodies. Furthermore, it is understood that methodsdescribed herein which utilize 8G2 can also utilize functional fragments(e.g., antigen-binding fragments) of 8G2, antibodies which have the sameor similar epitopic specificity as 8G2, and combinations thereof,optionally in combination with antibodies or fragments having anepitopic specificity which is not the same as or similar to 8G2;similarly, methods described as utilizing 1D9 can also utilizefunctional fragments of 1D9, antibodies which have the same or similarepitopic specificity as 1D9, and combinations thereof, optionally incombination with antibodies or fragments having an epitopic specificitywhich is not the same as or similar to 1D9. Antibodies of the presentinvention can be raised against an appropriate immunogen, such asisolated and/or recombinant mammalian CCR2 protein or portion thereof,or synthetic molecules, such as synthetic peptides. In a preferredembodiment, cells which express receptor, such as transfected cells, canbe used as immunogens or in a screen for antibody which binds receptor.

The antibodies of the present invention, and fragments thereof, areuseful in therapeutic, diagnostic and research applications as describedherein. The present invention encompasses an antibody or functionalportion thereof of the present invention (e.g., mAb 1D9 or 8G2, orantigen-binding fragments thereof) for use in therapy (includingprophylaxis) or diagnosis (e.g., of particular diseases or conditions asdescribed herein), and use of such antibodies or functional portionsthereof for the manufacture of a medicament for use in treatment ofdiseases or conditions as described herein.

Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed as described herein, or using othersuitable techniques. A variety of methods have been described (see e.g.,Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowskiet al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y.); Current Protocols In Molecular Biology, Vol. 2(Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley &Sons: New York, N.Y.), Chapter 11, (1991)). Generally, a hybridoma canbe produced by fusing a suitable immortal cell line (e.g., a myelomacell line such as SP2/0) with antibody producing cells. The antibodyproducing cell, preferably those of the spleen or lymph nodes, areobtained from animals immunized with the antigen of interest. The fusedcells (hybridomas) can be isolated using selective culture conditions,and cloned by limiting dilution. Cells which produce antibodies with thedesired binding properties can be selected by a suitable assay (e.g.,ELISA).

Other suitable methods of producing or isolating antibodies which bindCCR2, including human or artificial antibodies, can be used, including,for example, methods which select recombinant antibody (e.g., singlechain Fv or Fab) from a library, or which rely upon immunization oftransgenic animals (e.g., mice) capable of producing a repertoire ofhuman antibodies (see e.g., Jakobovits et al., Proc. Natl. Acad. Sci.USA, 90: 2551-2555 (1993); Jakobovits et al., Nature, 362: 255-258(1993); Lonberg et al., U.S. Pat. No. 5,545,806; Surani et al., U.S.Pat. No. 5,545,807).

Single chain antibodies, and chimeric, humanized or primatized(CDR-grafted) antibodies, as well as chimeric or CDR-grafted singlechain antibodies, and the like, comprising portions derived fromdifferent species, are also encompassed by the present invention and theterm “antibody”. The various portions of these antibodies can be joinedtogether chemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques. For example,nucleic acids encoding a chimeric or humanized chain can be expressed toproduce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss etal., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;Winter, European Patent No. 0,239,400 B1; and Queen et al., U.S. Pat.Nos. 5,585,089, 5,698,761 and 5,698,762. See also, Newman, R. et al.,BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, andLadner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science,242: 423-426 (1988)) regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments ofchimeric, humanized, primatized or single chain antibodies, can also beproduced. Functional fragments of the foregoing antibodies retain atleast one binding function and/or modulation function of the full-lengthantibody from which they are derived. Preferred functional fragmentsretain an antigen-binding function of a corresponding full-lengthantibody (e.g., the ability to bind a mammalian CCR2). Particularlypreferred functional fragments retain the ability to inhibit one or morefunctions characteristic of a mammalian CCR2, such as a bindingactivity, a signaling activity, and/or stimulation of a cellularresponse. For example, in one embodiment, a functional fragment caninhibit the interaction of CCR2 with one or more of its ligands (e.g.,MCP-1, MCP-2, MCP-3 and/or MCP-4) and/or can inhibit one or morereceptor-mediated functions, such as leukocyte trafficking, HIV entryinto cells, T cell activation, inflammatory mediator release and/orleukocyte degranulation.

For example, antibody fragments capable of binding to a mammalian CCR2receptor or portion thereof, including, but not limited to, Fv, Fab,Fab′ and F(ab′)₂ fragments are encompassed by the invention. Suchfragments can be produced by enzymatic cleavage or by recombinanttechniques, for example. For instance, papain or pepsin cleavage cangenerate Fab or F(ab′)₂ fragments, respectively. Antibodies can also beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons has been introduced upstream of the natural stopsite. For example, a chimeric gene encoding a F(ab′)₂ heavy chainportion can be designed to include DNA sequences encoding the CH₁ domainand hinge region of the heavy chain.

The present invention relates to a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for CCR2,comprising an antigen binding region of nonhuman origin (e.g., rodent)and at least a portion of an immunoglobulin of human origin (e.g., ahuman framework region, a human constant region or portion thereof). Inone embodiment, the humanized immunoglobulin includes an antigen bindingregion of nonhuman origin which binds CCR2 and a constant region derivedfrom a human constant region. In another embodiment, the humanizedimmunoglobulin which binds CCR2 comprises a complementarity determiningregion of nonhuman origin and a variable framework region of humanorigin, and optionally, a constant region of human origin. For example,the humanized immunoglobulin can comprise a heavy chain and a lightchain, wherein the light chain comprises a complementarity determiningregion derived from an antibody of nonhuman origin which binds CCR2 anda framework region derived from a light chain of human origin, and theheavy chain comprises a complementarity determining region derived froman antibody of nonhuman origin which binds CCR2 and a framework regionderived from a heavy chain of human origin.

In one embodiment, the humanized immunoglobulin can compete with murine1D9 or 8G2 monoclonal antibody for binding to human CCR2. In a preferredembodiment, the antigen-binding region of the humanized immunoglobulin(a) is derived from 1D9 monoclonal antibody (e.g., as in a humanizedimmunoglobulin comprising CDR1, CDR2 and CDR3 of the 1D9 light chainand/or CDR1, CDR2 and CDR3 of the 1D9 heavy chain) or (b) is derivedfrom 8G2 monoclonal antibody (e.g., as in a humanized immunoglobulincomprising CDR1, CDR2 and CDR3 of the 8G2 light chain and/or CDR1, CDR2and CDR3 of the 8G2 heavy chain). Chimeric or CDR-grafted single chainantibodies are also encompassed by the term humanized immunoglobulin.

The present invention also relates to a humanized immunoglobulin lightchain or antigen-binding fragment thereof or a humanized immunoglobulinheavy chain or antigen-binding fragment thereof. In one embodiment, theinvention relates to a humanized light chain comprising a light chainCDR (i.e., one or more CDRs) of nonhuman origin and a human light chainframework region. In another embodiment, the present invention relatesto a humanized immunoglobulin heavy chain comprising a heavy chain CDR(i.e., one or more CDRs) of nonhuman origin and a human heavy chainframework region. The CDRs can be derived from a nonhumanimmunoglobulin.

Naturally occurring immunoglobulins have a common core structure inwhich two identical light chains (about 24 kD) and two identical heavychains (about 55 or 70 kD) form a tetramer. The amino-terminal portionof each chain is known as the variable (V) region and can bedistinguished from the more conserved constant (C) regions of theremainder of each chain. Within the variable region of the light chainis a C-terminal portion known as the J region. Within the variableregion of the heavy chain, there is a D region in addition to the Jregion. Most of the amino acid sequence variation in immunoglobulins isconfined to three separate locations in the V regions known ashypervariable regions or complementarity determining regions (CDRs)which are directly involved in antigen binding. Proceeding from theamino-terminus, these regions are designated CDR1, CDR2 and CDR3,respectively. The CDRs are held in place by more conserved frameworkregions (FRs). Proceeding from the amino-terminus, these regions aredesignated FR1, FR2, FR3, and FR4, respectively. The locations of CDRand FR regions and a numbering system have been defined by Kabat et al.(Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, U.S. GovernmentPrinting Office (1991)).

Human immunoglobulins can be divided into classes and subclasses,depending on the isotype of the heavy chain. The classes include IgG,IgM, IgA, IgD and IgE, in which the heavy chains are of the gamma (γ),mu.(μ), alpha (α), delta (δ) or epsilon (ε) type, respectively.Subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, in which theheavy chains are of the γ1, γ2, γ3, γ4, α1 and α2 type, respectively.Human immunoglobulin molecules of a selected class or subclass maycontain either a kappa (κ) or lambda (λ) light chain. See e.g., Cellularand Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50,W. B. Saunders Co, Philadelphia, Pa. (1991); Nisonoff, A., Introductionto Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, SinauerAssociates, Inc., Sunderland, Mass. (1984).

The term “immunoglobulin” as used herein includes whole antibodies andbiologically functional fragments thereof. Such biologically functionalfragments retain at least one antigen-binding function of acorresponding full-length antibody (e.g., specificity for CCR2 ofantibody 1D9), and preferably, retain the ability to inhibit theinteraction of CCR2 with one or more of its ligands (e.g., HIV, MCP-1,MCP-2, MCP-3, MCP-4). Examples of biologically functional antibodyfragments which can be used include fragments capable of binding toCCR2, such as single chain antibodies, Fv, Fab, Fab′ and F(ab′)₂fragments. Such fragments can be produced by enzymatic cleavage or byrecombinant techniques. For instance, papain or pepsin cleavage can beused to generate Fab or F(ab′)₂ fragments, respectively. Antibodies canalso be produced in a variety of truncated forms using antibody genes inwhich one or more stop codons have been introduced upstream of thenatural stop site. For example, a chimeric gene encoding the heavy chainof an F(ab′)₂ fragment can be designed to include DNA sequences encodingthe CH₁ domain and hinge region of the heavy chain. As used herein, anantigen-binding fragment of a humanized immunoglobulin heavy or lightchain is intended to mean a fragment which binds to an antigen whenpaired with a complementary chain. That is, an antigen-binding fragmentof a humanized light chain will bind to an antigen when paired with aheavy chain (e.g., murine, chimeric, humanized) comprising a variableregion, and an antigen-binding fragment of a humanized heavy chain willbind to an antigen when paired with a light chain (e.g., murine,chimeric, humanized) comprising a variable region.

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of differentorigin, wherein at least one portion is of human origin. For example,the humanized antibody can comprise portions derived from animmunoglobulin of nonhuman origin with the requisite specificity, suchas a mouse, and from immunoglobulin sequences of human origin (e.g.,chimeric immunoglobulin), joined together chemically by conventionaltechniques (e.g., synthetic) or prepared as a contiguous polypeptideusing genetic engineering techniques (e.g., DNA encoding the proteinportions of the chimeric antibody can be expressed to produce acontiguous polypeptide chain). Another example of a humanizedimmunoglobulin of the present invention is an immunoglobulin containingone or more immunoglobulin chains comprising a CDR derived from anantibody of nonhuman origin and a framework region derived from a lightand/or heavy chain of human origin (e.g., CDR-grafted antibodies with orwithout framework changes). Chimeric or CDR-grafted single chainantibodies are also encompassed by the term humanized immunoglobulin.See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397;Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al.,WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1;Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400B1; Padlan, E. A. et al., European Patent Application No. 0,519,596 A1.See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No.5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)),regarding single chain antibodies.

For example, humanized immunoglobulins can be produced using syntheticand/or recombinant nucleic acids to prepare genes (e.g., cDNA) encodingthe desired humanized chain. For example, nucleic acid (e.g., DNA)sequences coding for humanized variable regions can be constructed usingPCR mutagenesis methods to alter DNA sequences encoding a human orhumanized chain, such as a DNA template from a previously humanizedvariable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17:5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993);Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Usingthese or other suitable methods, variants can also be readily produced.In one embodiment, cloned variable regions can be mutagenized, andsequences encoding variants with the desired specificity can be selected(e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No.5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

The antigen binding region of the humanized immunoglobulin (the nonhumanportion) can be derived from an immunoglobulin of nonhuman origin(referred to as a donor immunoglobulin) having binding specificity forCCR2. For example, a suitable antigen binding region can be derived fromthe murine monoclonal antibody 1D9. Other sources include CCR2-specificantibodies obtained from nonhuman sources, such as rodent (e.g., mouse,rat), rabbit, pig goat or non-human primate (e.g., monkey).Additionally, other polyclonal or monoclonal antibodies, such asantibodies which bind to the same or similar epitope as the 1D9antibody, can be made (e.g., Kohler et al., Nature, 256:495-497 (1975);Harlow et al., 1988, Antibodies: A Laboratory Manual, (Cold SpringHarbor, N.Y.); and Current Protocols in Molecular Biology, Vol. 2(Supplement 27, Summer '94), Ausubel et al., Eds. (John Wiley & Sons:New York, N.Y.), Chapter 11 (1991)).

For example, antibodies can be raised against an appropriate immunogenin a suitable mammal (e.g., a mouse, rat, rabbit or sheep). Cellsbearing CCR2, membrane fractions containing CCR2, and immunogenicfragments of CCR2 are examples of suitable immunogens.Antibody-producing cells (e.g., a lymphocyte) can be isolated from, forexample, the lymph nodes or spleen of an immunized animal. The cells canthen be fused to a suitable immortalized cell (e.g., a myeloma cellline), thereby forming a hybridoma. Fused cells can be isolatedemploying selective culturing techniques. Cells which produce antibodieswith the desired specificity can be selected by a suitable assay (e.g.,ELISA). Immunoglobulins of nonhuman origin having binding specificityfor CCR2 can also be obtained from antibody libraries (e.g., a phagelibrary comprising nonhuman Fab molecules).

In one embodiment, the antigen binding region of the humanizedimmunoglobulin comprises a CDR of nonhuman origin. In this embodiment,the humanized immunoglobulin having binding specificity for CCR2comprises at least one CDR of nonhuman origin. For example, CDRs can bederived from the light and heavy chain variable regions ofimmunoglobulins of nonhuman origin, such that a humanized immunoglobulinincludes substantially heavy chain CDR1, CDR2 and/or CDR3, and/or lightchain CDR1, CDR2 and/or CDR3, from one or more immunoglobulins ofnonhuman origin, and the resulting humanized immunoglobulin has bindingspecificity for CCR2. Preferably, all three CDRs of a selected chain aresubstantially the same as the CDRs of the corresponding chain of adonor, and more preferably, all three CDRs of the light and heavy chainsare substantially the same as the CDRs of the corresponding donor chain.In one embodiment, the invention relates to an immunoglobulin havingbinding specificity for CCR2 comprising a humanized light chain orantigen-binding fragment thereof comprising CDR1, CDR2 and CDR3 of thelight chain of the 1D9 antibody and a heavy chain, e.g., a human heavychain. The invention also includes an immunoglobulin having bindingspecificity for CCR2 comprising a humanized heavy chain orantigen-binding fragment thereof comprising CDR1, CDR2 and CDR3 of theheavy chain of the 1D9 antibody and a light chain, e.g., a human lightchain.

The invention also relates to an immunoglobulin having bindingspecificity for CCR2 comprising a light chain and a heavy chain, whereinthe light chain comprises at least 1 CDR of an antibody of non-humanorigin (e.g., 1D9) and framework and constant regions of human origin(e.g., SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15),and wherein the heavy chain comprises a variable region of non-humanorigin (e.g., from 1D9) and a constant region of human origin. Theinvention also provides antigen-binding fragments of theseimmunoglobulins. The invention also relates to an immunoglobulin havingbinding specificity for CCR2 comprising a light chain and a heavy chain,wherein the light chain comprises a variable chain of non-human origin(e.g., from 1D9) and a constant region of human origin, and wherein theheavy chain comprises at least 1 CDR of an antibody of non-human origin(e.g., 1D9) and framework and constant regions of human origin (e.g.,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20). Theinvention also provides antigen-binding fragments of theseimmunoglobulins.

The portion of the humanized immunoglobulin or immunoglobulin chainwhich is of human origin (the human portion) can be derived from anysuitable human immunoglobulin or immunoglobulin chain. For example, ahuman constant region or portion thereof, if present, can be derivedfrom the κ or λ light chains, and/or the γ (e.g., γ1, γ2, γ3, γ4), μ, α(e.g., α1, α2), δ or ε heavy chains of human antibodies, includingallelic variants. A particular constant region (e.g., IgG1), variant orportions thereof can be selected in order to tailor effector function.For example, a mutated constant region (variant) can be incorporatedinto a fusion protein to minimize binding to Fc receptors and/or abilityto fix complement (see e.g., Winter et al., GB 2,209,757 B; Morrison etal., WO 89/07142; Morgan et al, WO 94/29351, Dec. 22, 1994).

If present, human framework regions (e.g., of the light chain variableregion) are preferably derived from a human antibody variable regionhaving sequence similarity to the analogous or equivalent region (e.g.,light chain variable region) of the antigen binding region donor. Othersources of framework regions for portions of human origin of a humanizedimmunoglobulin include human variable consensus sequences (see, e.g.,Kettleborough, C. A. et al., Protein Engineering 4:773-783 (1991);Carter et al., WO 94/04679, published Mar. 3, 1994)). For example, thesequence of the antibody or variable region used to obtain the nonhumanportion can be compared to human sequences as described in Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, U.S. Government Printing Office(1991). In a particularly preferred embodiment, the framework regions ofa humanized immunoglobulin chain are derived from a human variableregion having at least about 60% overall sequence identity, preferablyat least about 70% overall sequence identity and more preferably atleast about 85% overall sequence identity, with the variable region ofthe nonhuman donor (e.g., murine antibody 1D9). A human portion can alsobe derived from a human antibody having at least about 65% sequenceidentity, and preferably at least about 70% sequence identity, withinthe particular portion (e.g., FR) being used, when compared to theequivalent portion (e.g., FR) of the nonhuman donor.

In one embodiment, the humanized immunoglobulin comprises at least oneof the framework regions (FR) derived from one or more chains of anantibody of human origin. Thus, the FR can include a FR1 and/or FR2and/or FR3 and/or FR4 derived from one or more antibodies of humanorigin. Preferably, the human portion of a selected humanized chainincludes FR1, FR2, FR3 and FR4 derived from a variable region of humanorigin (e.g., from a human immunoglobulin chain, from a human consensussequence).

The immunoglobulin portions of nonhuman and human origin for use in thepresent invention have sequences identical to immunoglobulins orimmunoglobulin portions from which they are derived or to variantsthereof. Such variants include mutants differing by the addition,deletion, or substitution of one or more residues. As indicated above,the CDRs which are of nonhuman origin are substantially the same as inthe nonhuman donor, and preferably are identical to the CDRs of thenonhuman donor. As described in Example 2, changes in the frameworkregion, such as those which substitute a residue of the framework regionof human origin with a residue from the corresponding position of thedonor, can be made. One or more mutations in the framework region can bemade, including deletions, insertions and substitutions of one or moreamino acids. Several such substitutions are described in the design ofhumanized 1D9 antibodies in Example 2. For a selected humanized antibodyor chain, framework mutations can be designed as described herein.Preferably, the humanized immunoglobulins can bind CCR2 with an affinitysimilar to or better than that of the nonhuman donor. Variants can beproduced by a variety of suitable methods, including mutagenesis ofnonhuman donor or acceptor human chains.

The humanized immunoglobulins of the present invention have bindingspecificity for human CCR2. In a preferred embodiment, the humanizedimmunoglobulin of the present invention has at least one functionalcharacteristic of murine antibody 1D9, such as binding function (e.g.,having specificity for CCR2, having the same or similar epitopicspecificity), and/or inhibitory function (e.g., the ability to inhibitCCR2-dependent function in vitro and/or in vivo, such as the ability toinhibit the binding of a cell bearing CCR2 to a ligand thereof (e.g., achemokine)). Thus, preferred humanized immunoglobulins can have thebinding specificity of the murine antibody 1D9, the epitopic specificityof murine antibody 1D9 (e.g., can compete with murine 1D9, a chimeric1D9 antibody, or humanized 1D9 for binding to CCR2 (e.g., on a cellbearing CCR2)), and/or inhibitory function of murine antibody 1D9.

The binding function of a humanized immunoglobulin having bindingspecificity for CCR2 can be detected by standard immunological methods,for example using assays which monitor formation of a complex betweenhumanized immunoglobulin and CCR2 (e.g., a membrane fraction comprisingCCR2, on a cell bearing CCR2, human cell line or recombinant host cellcomprising nucleic acid encoding CCR2 which expresses CCR2). Bindingand/or adhesion assays or other suitable methods can also be used inprocedures for the identification and/or isolation of humanizedimmunoglobulins (e.g., from a library) with the requisite specificity(e.g., an assay which monitors adhesion between a cell bearing CCR2 anda ligand thereof (e.g., HIV, MCP-1, MCP-2, MCP-3, MCP-4), or othersuitable methods.

The immunoglobulin portions of nonhuman and human origin for use in thepresent invention include light chains, heavy chains and portions oflight and heavy chains. These immunoglobulin portions can be obtained orderived from immunoglobulins (e.g., by de novo synthesis of a portion),or nucleic acid molecules encoding an immunoglobulin or chain thereofhaving the desired property (e.g., binding CCR2, sequence similarity)can be produced and expressed. Humanized immunoglobulins comprising thedesired portions (e.g., antigen binding region, CDR, FR, C region) ofhuman and nonhuman origin can be produced using synthetic and/orrecombinant nucleic acids to prepare genes (e.g., cDNA) encoding thedesired humanized chain. To prepare a portion of a chain, one or morestop codons can be introduced at the desired position. For example,nucleic acid (e.g., DNA) sequences coding for newly designed humanizedvariable regions can be constructed using PCR mutagenesis methods toalter existing DNA sequences (see e.g., Kamman, M., et al., Nucl. AcidsRes. 17:5404 (1989)). PCR primers coding for the new CDRs can behybridized to a DNA template of a previously humanized variable regionwhich is based on the same, or a very similar, human variable region(Sato, K., et al., Cancer Research 53:851-856 (1993)). If a similar DNAsequence is not available for use as a template, a nucleic acidcomprising a sequence encoding a variable region sequence can beconstructed from synthetic oligonucleotides (see e.g., Kolbinger, F.,Protein Engineering 8:971-980 (1993)). A sequence encoding a signalpeptide can also be incorporated into the nucleic acid (e.g., onsynthesis, upon insertion into a vector). If the natural signal peptidesequence is unavailable, a signal peptide sequence from another antibodycan be used (see, e.g., Kettleborough, C. A., Protein Engineering4:773-783 (1991)). Using these methods, methods described herein orother suitable methods, variants can be readily produced. In oneembodiment, cloned variable regions can be mutagenized, and sequencesencoding variants with the desired specificity can be selected (e.g.,from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

The invention relates to a humanized immunoglobulin light chain orantigen-binding fragment thereof, said light chain or antigen-bindingfragment thereof having an amino acid sequence comprising at least afunctional portion of the light chain variable region amino acidsequence of SEQ ID NO: 9. In a preferred embodiment, the amino acidsequence comprises at least one, preferably two, and more preferablythree of the CDRs of SEQ ID NO: 9. The invention also relates to ahumanized immunoglobulin heavy chain or antigen-binding fragmentthereof, said heavy chain or antigen-binding fragment thereof having anamino acid sequence comprising at least a functional portion of theheavy chain variable region amino acid sequence shown in SEQ ID NO: 10.In a preferred embodiment, the amino acid sequence comprises at leastone, preferably two, and more preferably three of the CDRs of SEQ ID NO:10. It is noted that all murine sequences described herein are derivedfrom Mus musculus.

According to one embodiment of the invention, a humanized immunoglobulinlight chain or antigen-binding fragment thereof having bindingspecificity for CCR2 can comprise an amino acid sequence selected fromthe group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, andSEQ ID NO: 15. According to another embodiment of the invention, ahumanized immunoglobulin heavy chain or antigen-binding fragment thereofhaving binding specificity for CCR2 can comprise an amino acid sequenceselected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19, and SEQ ID NO: 20. In a particular embodiment, a humanizedimmunoglobulin of the invention can comprise both a light chain orantigen-binding fragment thereof having binding specificity for CCR2,comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, and aheavy chain or antigen-binding fragment thereof having bindingspecificity for CCR2 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQID NO: 20. In one embodiment, the humanized immunoglobulin light chainor antigen-binding fragment thereof having binding specificity for CCR2can be encoded by a nucleic acid molecule comprising SEQ ID NO: 98. Inanother embodiment, the humanized immunoglobulin heavy chain orantigen-binding fragment thereof having binding specificity for CCR2 canbe encoded by a nucleic acid molecule comprising SEQ ID NO: 97.

The invention also relates to a chimeric immunoglobulin orantigen-binding fragment thereof having binding specificity for CCR2comprising a light chain variable region of nonhuman origin and a humanconstant region (e.g., a light chain constant region). The inventionfurther relates to a chimeric immunoglobulin or antigen-binding fragmentthereof having binding specificity for CCR2 comprising a heavy chainvariable region of nonhuman origin and a human constant region (e.g., aheavy chain constant region). In another embodiment, the chimericimmunoglobulin or antigen-binding fragment thereof having bindingspecificity for CCR2 comprises a light chain variable chain region ofnonhuman origin and a heavy chain variable region of nonhuman origin andfurther comprises a human constant region (e.g., a human light chainconstant region and/or a human heavy chain constant region).

Nucleic Acids and Constructs

The present invention also relates to isolated and/or recombinant(including, e.g., essentially pure) nucleic acid molecules comprisingnucleic acid sequences which encode a humanized immunoglobulin orhumanized immunoglobulin light or heavy chain of the present invention.

Nucleic acid molecules referred to herein as “isolated” are nucleic acidmolecules which have been separated away from the nucleic acids of thegenomic DNA or cellular RNA of their source of origin (e.g., as itexists in cells or in a mixture of nucleic acids such as a library), andinclude nucleic acid molecules obtained by methods described herein orother suitable methods, including essentially pure nucleic acidmolecules, nucleic acid molecules produced by chemical synthesis, bycombinations of biological and chemical methods, and recombinant nucleicacid molecules which are isolated (see e.g., Daugherty, B. L. et al.,Nucleic Acids Res., 19(9): 2471-2476 (1991); Lewis, A. P. and J. S.Crowe, Gene, 101: 297-302 (1991)).

Nucleic acid molecules referred to herein as “recombinant” are nucleicacid molecules which have been produced by recombinant DNA methodology,including those nucleic acid molecules that are generated by procedureswhich rely upon a method of artificial recombination, such as thepolymerase chain reaction (PCR) and/or cloning into a vector usingrestriction enzymes. “Recombinant” nucleic acid molecules are also thosethat result from recombination events that occur through the naturalmechanisms of cells, but are selected for after the introduction to thecells of nucleic acids designed to allow and make probable a desiredrecombination event.

The present invention also relates more specifically to isolated and/orrecombinant nucleic acid molecules comprising a nucleotide sequencewhich encodes a humanized 1D9 immunoglobulin (i.e., a humanizedimmunoglobulin of the present invention in which the nonhuman portion isderived from the murine monoclonal antibody 1D9) or chain thereof. Inone embodiment, the light chain comprises three complementaritydetermining regions derived from the light chain of the 1D9 antibody,and the heavy chain comprises three complementarity determining regionsderived from the heavy chain of the 1D9 antibody. Such nucleic acidmolecules include, for example, (a) a nucleic acid molecule comprising asequence which encodes a polypeptide comprising the amino acid sequenceof the heavy chain variable region of a humanized 1D9 immunoglobulin(e.g., heavy chain variable region of FIGS. 8 and 21) (e.g., nucleotides58411 of SEQ ID NO: 96); (b) a nucleic acid molecule comprising asequence which encodes a polypeptide comprising the amino acid sequenceof the light chain variable region of a humanized 1D9 immunoglobulin(e.g., light chain variable region of FIGS. 7 and 22) (e.g., nucleotides52-390 of SEQ ID NO: 95); (c) a nucleic acid molecule comprising asequence which encodes at least a functional portion of the light orheavy chain variable region of a humanized 1D9 immunoglobulin (e.g., aportion sufficient for antigen binding of a humanized immunoglobulinwhich comprises said chain). Due to the degeneracy of the genetic code,a variety of nucleic acids can be made which encode a selectedpolypeptide. In one embodiment, the nucleic acid comprises thenucleotide sequence of the variable region as set forth or substantiallyas set forth in FIG. 21 or as set forth or substantially as set forth inFIG. 22, including double or single-stranded polynucleotides. (Althoughvarious figures may illustrate polypeptides which are larger than thevariable region (i.e., include a signal peptide coding sequence or aportion of a constant region coding sequence), reference to the variableregion of a particular figure is meant to include the variable regionportion of the sequence shown). Isolated and/or recombinant nucleic acidmolecules meeting these criteria can comprise nucleic acid moleculesencoding sequences identical to sequences of humanized 1D9 antibody orvariants thereof as discussed above.

Nucleic acid molecules of the present invention can be used in theproduction of humanized immunoglobulins having binding specificity forCCR2. For example, a nucleic acid molecule (e.g., DNA) encoding ahumanized immunoglobulin of the present invention can be incorporatedinto a suitable construct (e.g., a vector) for further manipulation ofsequences or for production of the encoded polypeptide in suitable hostcells.

Targeting Molecules

The invention also relates to targeting molecules which can effectuatethe interaction of a CCR2-expressing cell with a target cell. Thetargeting molecule includes a first binding moiety which can bindmammalian CCR2, and a second binding moiety which can bind a moleculeexpressed on the surface of a target cell. Preferred target cellsinclude tumor cells and virus infected cells. A variety of moleculeswhich are expressed at higher levels or uniquely on tumor cells (e.g.,tumor antigens, such as Lewis Y, HER-2/neu, disialoganglioside G3,carcinoembrionic antigen, CD30) and/or virus infected cells (e.g., viralantigens, such as influenza virus hemagglutinin, Epstein-Barr virusLMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, HIV gp120) areknown in the art. The targeting molecule can contain any suitablebinding second moiety which binds to a molecule expressed on a desiredtarget cell (see, for example Ring, U.S. Pat. No. 5,948,647, the entireteachings of which are incorporated herein by reference). Suitablebinding moieties include, for example, proteins and peptides (includingpost-translationally modified forms e.g., glycosylated, phosphorylated,lipidated), sugars, lipids, peptidomimetics, small organic molecules,nucleic acids and other agents which bind mammalian CCR2 or a moleculeexpressed on the surface of a target cell. Suitable binding moieties canbe identified using any suitable method, such as the binding assaysdescribed herein.

In a preferred embodiment, the first binding moiety can be, for example,a humanized immunoglobulin of the invention which binds mammalian CCR2or antigen-binding fragment thereof (e.g., Fab, Fv, Fab′, F(ab)′₂). Thesecond binding moiety can be, for example, an antibody (e.g., a secondhumanized immunoglobulin) or antigen-binding fragment thereof whichbinds to a molecule expressed on the target cell or antigen bindingfragment thereof. Where the targeting molecule comprises a first bindingmoiety which is a humanized anti-CCR2 immunoglobulin or antigen-bindingfragment thereof, it is preferred that the humanized anti-CCR2immunoglobulin does not inhibit binding of ligand to CCR2.

The first binding moiety can be directly or indirectly bonded to thesecond binding moiety through a variety of suitable linkages. Forexample, when the first binding moiety and the second binding moiety areboth proteins or peptides, the moieties can be part of a contiguouspolypeptide (i.e., a fusion protein). Where the targeting molecule is afusion protein, the first and second binding moieties can be arranged onthe polypeptide in any suitable configuration. The first and secondbinding moieties can be indirectly bonded through a (i.e., one or more)peptide linker, or bonded directly to each other through a peptide bond.

Where the binding moieties are not part of a contiguous polypeptide theycan be directly bonded by a chemical bond formed by reaction of afunctional group (or activated derivative thereof) on the first moietywith a second functional group (or activated derivative thereof) on thesecond moiety. For example, two thiols can react to form a disulfidebond and an amine can react with a carboxylic acid or acyl halide toform an amide. A variety of other suitable reactions which can be usedare known in the art (see, for example, Hermanson, G. T., BioconjugateTechniques, Academic Press: San Diego, Calif. (1996)). The bindingmoieties can be indirectly bonded through a suitable linker (e.g., apeptide linker). Generally, a linker contains two reactive groups whichcan react to form bonds with the first binding moiety and/or the secondbinding moiety. Linkers which contain two different reactive groups(e.g., a heterobifunctional linker) can be used to selectively conjugatethe first binding moiety to the second binding moiety. Many linkerswhich are suitable for forming conjugates between proteins, nucleicacids, peptides, vitamins, sugars, lipids, small organic molecules andother suitable agents are known (see, for example, U.S. Pat. Nos.5,856,571, 5,880,270; Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)).

Preferably, the independent activities of the binding moieties (e.g.,binding activities, chemoattractant activity) of the targeting moleculeare not significantly different from the activities of the bindingmoieties as separate molecular entities. For example, where the firstbinding moiety is a humanized immunoglobulin or antigen-binding fragmentthat binds CCR2, the targeting molecule can bind to CCR2 with anaffinity which is within a factor of about 1000, preferably within afactor of 100, more preferably within a factor of 10 or substantiallythe same as the affinity of the free antibody or antigen-bindingfragment. Target molecules with these preferred characteristics can beprepared using any suitable method. The resulting targeting molecule canthen be assayed for binding (e.g., by ELISA) and for chemoattractantactivity.

In one embodiment, the targeting molecule is a bispecific humanizedantibody or bispecific antigen-binding fragment thereof (e.g., F(ab′)₂)which has specificity for mammalian CCR2 and a molecule expressed on atarget cell (e.g., tumor antigen, viral antigen). Bispecific antibodiescan be secreted by triomas and hybrid hybridomas. The supernatants oftriomas and hybrid hybridomas can be assayed for bispecific antibodyusing a suitable assay (e.g., ELISA), and bispecific antibodies can bepurified using conventional methods. These antibodies can then behumanized according to methods described herein. Thus, the inventionprovides a targeting molecule which is a humanized bispecific antibodyhaving binding specificity for CCR2 and an antigen expressed on a targetcell, or a bivalent antigen-binding fragment of the bispecific antibody.The invention also relates to a method of effectuating the interactionof a CCR2-bearing cell with a target cell in a patient, comprisingadministering to the patient an effective amount of a targeting moleculewhich is a humanized bispecific antibody having binding specificity forCCR2 and an antigen expressed on a target cell, or a bivalentantigen-binding fragment of the bispecific antibody.

Method of Producing Humanized Immunoglobulins Having Specificity forCCR2

Another aspect of the invention relates to a method of preparing ahumanized immunoglobulin which has binding specificity for CCR2. Thehumanized immunoglobulin can be obtained, for example, by the expressionof one or more recombinant nucleic acids encoding a humanizedimmunoglobulin having binding specificity for CCR2 in a suitable hostcell, for example.

Constructs or expression vectors suitable for the expression of ahumanized immunoglobulin having binding specificity for CCR2 are alsoprovided. The constructs can be introduced into a suitable host cell,and cells which express a humanized immunoglobulin of the presentinvention can be produced and maintained in culture. Suitable host cellscan be prokaryotic, including bacterial cells such as E. coli, B.subtilis and or other suitable bacteria, or eucaryotic, such as fungalor yeast cells (e.g., Pichia pastoris, Aspergillus species,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa),or other lower eucaryotic cells, and cells of higher eucaryotes such asthose from insects (e.g., Sf9 insect cells (WO 94/26087, O'Connor,published Nov. 24, 1994)) or mammals (e.g., COS cells, such as COS-1(ATCC Accession No. CRL-1650) and COS-7 (ATCC Accession No. CRL-1651),CHO (e.g., ATCC Accession No. CRL-9096), 293 (ATCC Accession No.CRL-1573), HeLa (ATCC Accession No. CCL-2), CV1 (ATCC Accession No.CCL-70), WOP (Dailey et al., J. Virol. 54:739-749 (1985)), 3T3, 293T(Pear et al., Proc. Natl. Acad. Sci. U.S.A., 90:8392-8396 (1993)), NSOcells, SP2/0, HuT 78 cells, and the like (see, e.g., Ausubel, F. M. etal., eds. Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons Inc., (1993)).

Host cells which produce a humanized immunoglobulin having bindingspecificity for CCR2 can be produced as follows. For example, a nucleicacid encoding all or part of the coding sequence for the desiredhumanized immunoglobulin can be inserted into a nucleic acid vector,e.g., a DNA vector, such as a plasmid, virus or other suitable repliconfor expression. A variety of vectors are available, including vectorswhich are maintained in single copy or multiple copy, or which becomeintegrated into the host cell chromosome.

Suitable expression vectors can contain a number of components,including, but not limited to one or more of the following: an origin ofreplication; a selectable marker gene; one or more expression controlelements, such as a transcriptional control element (e.g., a promoter,an enhancer, terminator), and/or one or more translation signals; asignal sequence or leader sequence for membrane targeting or secretion.In a construct, a signal sequence can be provided by the vector or othersource. For example, the transcriptional and/or translational signals ofan immunoglobulin can be used to direct expression.

A promoter can be provided for expression in a suitable host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding a humanized immunoglobulinor immunoglobulin chain, such that it directs expression of the encodedpolypeptide. A variety of suitable promoters for procaryotic (e.g., lac,tac, T3, T7 promoters for E. coli) and eucaryotic (e.g., yeast alcoholdehydrogenase (ADH1), SV40, CMV) hosts are available.

In addition, the expression vectors typically comprise a selectablemarker for selection of host cells carrying the vector, and, in the caseof replicable expression vector, an origin or replication. Genesencoding products which confer antibiotic or drug resistance are commonselectable markers and may be used in procaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene for tetracycline resistance) andeucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated. In one embodiment, thevector is pLKTOK38. The present invention also relates to cells carryingthese expression vectors. An expression vector comprising a fused geneencoding a humanized immunoglobulin light chain, said gene comprising anucleotide sequence encoding a CDR derived from a light chain of anonhuman antibody having binding specificity for CCR2 and a frameworkregion derived from a light chain of human origin.

Thus, the invention includes an expression vector comprising a geneencoding a humanized immunoglobulin light chain, said gene comprising anucleotide sequence encoding a CDR derived from a light chain of anonhuman antibody having binding specificity for CCR2 and a frameworkregion derived from a light chain of human origin. The invention alsorelates to an expression vector comprising a gene encoding a humanizedimmunoglobulin heavy chain, said gene comprising a nucleotide sequenceencoding a CDR derived from a heavy chain of a nonhuman antibody havingbinding specificity for CCR2 and a framework region derived from a heavychain of human origin. In on embodiment, the nonhuman antibody is murineantibody 1D9. The invention also includes host cells comprising theexpression vectors of the invention. The invention also relates to anisolated or recombinant gene encoding a humanized immunoglobulin lightor heavy chain comprising a first nucleic acid sequence encoding anantigen binding region derived from murine monoclonal antibody 1D9; anda second nucleic acid sequence encoding at least a portion of a constantregion of an immunoglobulin of human origin.

The invention also relates to a host cell (e.g., which expresses ahumanized immunoglobulin or an antigen binding fragment thereof havingspecificity for CCR2) comprising a first recombinant nucleic acidmolecule encoding a humanized immunoglobulin light chain or fragmentthereof and a second recombinant nucleic acid molecule encoding ahumanized immunoglobulin heavy chain or fragment thereof, wherein saidfirst nucleic acid molecule comprises a nucleotide sequence encoding aCDR derived from the light chain of murine antibody 1D9 and a frameworkregion derived from a light chain of human origin, and wherein saidsecond nucleic acid molecule comprises a nucleotide sequence encoding aCDR derived from the heavy chain of murine antibody 1D9 and a frameworkregion derived from a heavy chain of human origin. The invention alsoincludes a method of preparing a humanized immunoglobulin orantigen-binding fragment thereof comprising maintaining a host cell ofthe invention under conditions appropriate for expression of a humanizedimmunoglobulin, whereby humanized immunoglobulin chains are expressedand a humanized immunoglobulin or antigen-binding fragment thereofhaving specificity for CCR2 is produced. The method can further comprisethe step of isolating the humanized immunoglobulin or fragment thereof.

For example, a nucleic acid molecule (i.e., one or more nucleic acidmolecules) encoding the heavy and light chains of a humanizedimmunoglobulin having binding specificity for CCR2, or a construct(i.e., one or more constructs) comprising such nucleic acid molecule(s),can be introduced into a suitable host cell by a method appropriate tothe host cell selected (e.g., transformation, transfection,electroporation, infection), such that the nucleic acid molecule(s) areoperably linked to one or more expression control elements (e.g., in avector, in a construct created by processes in the cell, integrated intothe host cell genome). Host cells can be maintained under conditionssuitable for expression (e.g., in the presence of inducer, suitablemedia supplemented with appropriate salts, growth factors, antibiotic,nutritional supplements, etc.), whereby the encoded polypeptide(s) areproduced. If desired, the encoded protein (e.g., humanized 1D9 antibody)can be isolated from, e.g., the host cells, medium, milk. This processencompasses expression in a host cell of a transgenic animal (see e.g.,WO 92/03918, GenPharm International, published Mar. 19, 1992).

Fusion proteins can be produced in which a humanized immunoglobulin orimmunoglobulin chain is linked to a non-immunoglobulin moiety (i.e., amoiety which does not occur in immunoglobulins as found in nature) in anN-terminal location, C-terminal location or internal to the fusionprotein. For example, some embodiments can be produced by the insertionof a nucleic acid encoding immunoglobulin sequences into a suitableexpression vector, such as a pET vector (e.g., pET-15b, Novagen), aphage vector (e.g., pCANTAB 5 E, Pharmacia), or other vector (e.g.,pRIT2T Protein A fusion vector, Pharmacia). The resulting construct canbe introduced into a suitable host cell for expression. Upon expression,some fusion proteins can be isolated or purified from a cell lysate bymeans of a suitable affinity matrix (see e.g., Current Protocols inMolecular Biology (Ausubel, F. M. et al., eds., Vol. 2, Suppl. 26, pp.16.4.1-16.7.8 (1991)).

Therapeutic Methods and Compositions

The present invention provides humanized immunoglobulins which (1) canbind CCR2 in vitro and/or in vivo; and/or (2) can modulate an activityor function of CCR2, such as (a) binding function (e.g., the ability ofCCR2 to bind to a ligand) and/or (b) leukocyte trafficking, includingrecruitment and/or accumulation of leukocytes in tissues. Preferably thehumanized immunoglobulins are capable of selectively binding CCR2 invitro and/or in vivo, and inhibiting CCR2-mediated interactions. In oneembodiment, a humanized immunoglobulin can bind CCR2, and can inhibitbinding of CCR2 to one or more of its ligands (e.g., HIV, MCP-1, MCP-2,MCP-3, MCP-4).

The humanized immunoglobulins of the present invention are useful in avariety of processes with applications in research, diagnosis andtherapy. For instance, they can be used to detect, isolate, and/orpurify CCR2 or variants thereof (e.g., by affinity purification or othersuitable methods), and to study CCR2 structure (e.g., conformation) andfunction. The humanized immunoglobulins of the present invention canalso be used in diagnostic applications (e.g., in vitro, ex vivo) or tomodulate CCR2 function in therapeutic (including prophylactic)applications.

For example, the humanized immunoglobulins of the present invention canbe used to detect and/or measure the level of CCR2 in a sample (e.g.,tissues or body fluids, such as an inflammatory exudate, blood, serum,bowel fluid, on cells bearing CCR2). For example, a sample (e.g., tissueand/or body fluid) can be obtained from an individual and a suitableimmunological method can be used to detect and/or measure CCR2expression, including methods such as enzyme-linked immunosorbent assays(ELISA), including chemiluminescence assays, radioimmunoassay, andimmunohistology. In one embodiment, a method of detecting a selectedCCR2 in a sample is provided, comprising contacting a sample with ahumanized immunoglobulin of the present invention under conditionssuitable for specific binding of the humanized immunoglobulin to CCR2and detecting antibody-CCR2 complexes which are formed. In anapplication of the method, humanized immunoglobulins can be used toanalyze normal versus inflamed tissues (e.g., from a human) for CCR2reactivity and/or expression (e.g., immunohistologically)), to detectassociations between particular conditions and increased expression ofCCR2 (e.g., in affected tissues). The humanized immunoglobulins of thepresent invention permit immunological methods of assessment of thepresence of CCR2 in normal versus inflamed tissues, through which thepresence of disease, disease progress and/or the efficacy of anti-CCR2integrin therapy in inflammatory disease can be assessed.

The humanized immunoglobulins of the present invention can also be usedto modulate (e.g., inhibit (reduce or prevent)) binding function and/orleukocyte (e.g., lymphocyte, monocyte) trafficking modulated by CCR2.For example, humanized immunoglobulins which inhibit the binding of CCR2to a ligand (i.e., one or more ligands) can be administered according tothe method in the treatment of diseases associated with leukocyte (e.g.,lymphocyte, monocyte) infiltration of tissues. Additionally, humanizedimmunoglobulins which inhibit the binding of CCR2 to a ligand (i.e., oneor more ligands) can be administered according to the method in thetreatment of HIV. An effective amount of a humanized immunoglobulin ofthe present invention (i.e., one or more) is administered to anindividual (e.g., a mammal, such as a human or other primate) in orderto treat such a disease.

The humanized immunoglobulin is administered in an effective amountwhich inhibits binding of CCR2 to a ligand thereof. For therapy, aneffective amount will be sufficient to achieve the desired therapeutic(including prophylactic) effect (such as an amount sufficient to reduceor prevent CCR2-mediated binding and/or signalling). The humanizedimmunoglobulin can be administered in a single dose or multiple doses.The dosage can be determined by methods known in the art and can bedependent, for example, upon the individual's age, sensitivity,tolerance and overall well-being. Suitable dosages for antibodies can befrom about 0.1 mg/kg body weight to about 10.0 mg/kg body weight pertreatment.

According to the method, the humanized immunoglobulin can beadministered to an individual (e.g., a human) alone or in conjunctionwith another agent. A humanized immunoglobulin can be administeredbefore, along with or subsequent to administration of the additionalagent. Thus, the invention includes pharmaceutical compositionscomprising a humanized immunoglobulin or antigen-binding fragmentthereof of the invention and a suitable carrier. In one embodiment, morethan one humanized immunoglobulin which inhibits the binding of CCR2 toits ligands is administered. In another embodiment an additionalmonoclonal antibody is administered in addition to a humanizedimmunoglobulin of the present invention. In yet another embodiment, anadditional pharmacologically active ingredient (e.g., anantiinflammatory compound, such as sulfasalazine, another non-steroidalantiinflammatory compound, or a steroidal antiinflammatory compound) canbe administered in conjunction with a humanized immunoglobulin of thepresent invention.

A variety of routes of administration are possible, including, but notnecessarily limited to, parenteral (e.g., intravenous, intraarterial,intramuscular, subcutaneous injection), oral (e.g., dietary), topical,inhalation (e.g., intrabronchial, intranasal or oral inhalation,intranasal drops), or rectal, depending on the disease or condition tobe treated. Parenteral administration is a preferred mode ofadministration.

Formulation will vary according to the route of administration selected(e.g., solution, emulsion). An appropriate composition comprising thehumanized antibody to be administered can be prepared in aphysiologically acceptable vehicle or carrier. For solutions oremulsions, suitable carriers include, for example, aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles can include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles can include variousadditives, preservatives, or fluid, nutrient or electrolyte replenishers(See, generally, Remington's Pharmaceutical Sciences, 17th Edition, MackPublishing Co., PA, 1985). For inhalation, the compound can besolubilized and loaded into a suitable dispenser for administration(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

Thus, the invention includes a method of inhibiting HIV infection of acell, comprising contacting a cell with an effective amount of acomposition comprising a humanized immunoglobulin or antigen-bindingfragment thereof of the invention. The invention also relates to amethod of treating HIV or inhibiting HIV infection in a patientcomprising administering to the patient a composition comprising aneffective amount of a humanized immunoglobulin of or antigen-bindingfragment thereof of the invention.

The invention also relates to a method of inhibiting a functionassociated with binding of a chemokine to mammalian CCR2 or a functionalportion of CCR2, comprising contacting a composition comprising CCR2 orportion thereof with an effective amount of a humanized immunoglobulinor antigen-binding fragment thereof of the invention, wherein saidhumanized immunoglobulin inhibits binding of the chemokine to mammalianCCR2 and inhibits one or more functions associated with binding of thechemokine to CCR2. For example, the chemokine can be selected from thegroup consisting of MCP-1, MCP-2, MCP-3, MCP-4 and combinations thereof.

The invention also relates to a method of inhibiting leukocytetrafficking in a patient, comprising administering to the patient acomposition comprising an effective amount of a humanized immunoglobulinor antigen-binding fragment thereof of the invention which binds tomammalian CCR2 and inhibits binding of a ligand to the receptor. Forexample, the ligand can be a chemokine (e.g., MCP-1, MCP-2, MCP-3,MCP-4) or HIV.

The invention also relates to a method of inhibiting the interaction ofa first cell expressing CCR2 with a ligand (e.g., on a second cellexpressing a ligand of CCR2), comprising contacting the first cell withan effective amount of a humanized immunoglobulin or antigen-bindingfragment thereof of the invention, particularly wherein saidimmunoglobulin or fragment inhibits the binding of ligand to CCR2. Forexample, the cell can be selected from the group consisting oflymphocytes, monocytes, granulocytes, T cells, basophils, and cellscomprising a recombinant nucleic acid encoding CCR2 or a portionthereof. In one embodiment, the ligand is a chemokine (e.g., MCP-1,MCP-2, MCP-3, MCP-4). In another embodiment, the ligand is HIV.

The invention also includes a method of treating a CCR2-mediateddisorder in a patient, comprising administering to the patient aneffective amount of a humanized immunoglobulin or antigen-bindingfragment thereof of the invention which binds to mammalian CCR2. Thedisorder can include, but is not limited to, allergy, atherogenesis,anaphylaxis, malignancy, chronic and acute inflammatory disorders,histamine and IgE-mediated allergic reactions, shock, and rheumatoidarthritis, atherosclerosis, multiple sclerosis, stenosis, restenosis,allograft rejection, fibrotic disease, asthma, and inflammatoryglomerulopathies.

In a particular embodiment, the invention relates to a method ofinhibiting restenosis in a patient, comprising administering to thepatient an effective amount of a humanized immunoglobulin orantigen-binding fragment thereof of the invention which binds tomammalian CCR2. The invention also includes a humanized immunoglobulinor antigen-binding fragment thereof of the invention for use in therapyor diagnosis or for use in treating a CCR2-mediated disease or disorder.The invention also includes the use of a humanized immunoglobulin orantigen-binding fragment thereof of the invention for the manufacture ofa medicament for treating a CCR2-mediated disease.

Anti-idiotypic antibodies are also provided. Anti-idiotypic antibodiesrecognize antigenic determinants associated with the antigen-bindingsite of another antibody. Anti-idiotypic antibodies can be preparedagainst second antibody by immunizing an animal of the same species, andpreferably of the same strain, as the animal used to produce the secondantibody. See e.g., U.S. Pat. No. 4,699,880.

The present invention also pertains to the hybridoma cell linesdeposited under ATCC Accession Nos. HB-12549 and HB-12550, as well as tothe monoclonal antibodies produced by the hybridoma cell lines depositedunder ATCC Accession Nos. HB-12549 and HB-12550. The cell lines of thepresent invention have uses other than for the production of themonoclonal antibodies. For example, the cell lines of the presentinvention can be fused with other cells (such as suitably drug-markedhuman myeloma, mouse myeloma, human-mouse heteromyeloma or humanlymphoblastoid cells) to produce additional hybridomas, and thus providefor the transfer of the genes encoding the monoclonal antibodies. Inaddition, the cell lines can be used as a source of nucleic acidsencoding the anti-CCR2 immunoglobulin chains, which can be isolated andexpressed (e.g., upon transfer to other cells using any suitabletechnique (see e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Winter,U.S. Pat. No. 5,225,539)). For instance, clones comprising a rearrangedanti-CCR2 light or heavy chain can be isolated (e.g., by PCR) or cDNAlibraries can be prepared from mRNA isolated from the cell lines, andcDNA clones encoding an anti-CCR2 immunoglobulin chain can be isolated.Thus, nucleic acids encoding the heavy and/or light chains of theantibodies or portions thereof can be obtained and used in accordancewith recombinant DNA techniques for the production of the specificimmunoglobulin, immunoglobulin chain, or variants thereof (e.g.,humanized immunoglobulins) in a variety of host cells or in an in vitrotranslation system. For example, the nucleic acids, including cDNAs, orderivatives thereof encoding variants such as a humanized immunoglobulinor immunoglobulin chain, can be placed into suitable prokaryotic oreukaryotic vectors (e.g., expression vectors) and introduced into asuitable host cell by an appropriate method (e.g., transformation,transfection, electroporation, infection), such that the nucleic acid isoperably linked to one or more expression control elements (e.g., in thevector or integrated into the host cell genome). For production, hostcells can be maintained under conditions suitable for expression (e.g.,in the presence of inducer, suitable media supplemented with appropriatesalts, growth factors, antibiotic, nutritional supplements, etc.),whereby the encoded polypeptide is produced. If desired, the encodedprotein can be recovered and/or isolated (e.g., from the host cells,medium, milk). It will be appreciated that the method of productionencompasses expression in a host cell of a transgenic animal (see e.g.,WO 92/03918, GenPharm International, published Mar. 19, 1992).

As described herein, antibodies and functional fragments thereof of thepresent invention can block (inhibit) binding of a ligand to CCR2 and/orinhibit function associated with binding of the ligand to the CCR2. Asdiscussed below various methods can be used to assess inhibition ofbinding of a ligand to CCR2 and/or function associated with binding ofthe ligand to the receptor.

Binding Assays

As used herein “mammalian CCR2 protein” refers to naturally occurring orendogenous mammalian CCR2 proteins and to proteins having an amino acidsequence which is the same as that of a naturally occurring orendogenous corresponding mammalian CCR2 protein (e.g., recombinantproteins). Accordingly, as defined herein, the term includes maturereceptor protein, polymorphic or allelic variants, and other isoforms ofa mammalian CCR2 (e.g., produced by alternative splicing or othercellular processes), and modified or unmodified forms of the foregoing(e.g., glycosylated, unglycosylated). Mammalian CCR2 proteins can beisolated and/or recombinant proteins (including synthetically producedproteins). Naturally occurring or endogenous mammalian CCR2 proteinsinclude wild type proteins such as mature CCR2, polymorphic or allelicvariants and other isoforms which occur naturally in mammals (e.g.,humans, non-human primates), such as the CCR2a and CCR2b forms of thereceptor protein which are produced by alternative splicing of thecarboxy-terminus of the protein. Such proteins can be recovered orisolated from a source which naturally produces mammalian CCR2, forexample. These proteins and mammalian CCR2 proteins having the sameamino acid sequence as a naturally occurring or endogenous correspondingmammalian CCR2, are referred to by the name of the corresponding mammal.For example, where the corresponding mammal is a human, the protein isdesignated as a human CCR2 protein (e.g., a recombinant human CCR2produced in a suitable host cell).

“Functional variants” of mammalian CCR2 proteins include functionalfragments, functional mutant proteins, and/or functional fusion proteins(e.g., produced via mutagenesis and/or recombinant techniques).Generally, fragments or portions of mammalian CCR2 proteins includethose having a deletion (i.e., one or more deletions) of an amino acid(i.e., one or more amino acids) relative to the mature mammalian CCR2protein (such as N-terminal, C-terminal or internal deletions).Fragments or portions in which only contiguous amino acids have beendeleted or in which non-contiguous amino acids have been deletedrelative to mature mammalian CCR2 protein are also envisioned.

Generally, mutants of mammalian CCR2 proteins include natural orartificial variants of a mammalian CCR2 protein differing by theaddition, deletion and/or substitution of one or more contiguous ornon-contiguous amino acid residues (e.g., receptor chimeras). Suchmutations can be in a conserved region or nonconserved region (comparedto other CXC and/or CC chemokine receptors), extracellular, cytoplasmic,or transmembrane region, for example.

Generally, fusion proteins encompass polypeptides comprising a mammalianCCR2 (e.g., human CCR2) as a first moiety, linked via a peptide cond toa second moiety not occurring in the mammalian CCR2 as found in nature.Thus, the second moiety can be an amino acid, oligopeptide orpolypeptide. The first moiety can be in an N-terminal location,C-terminal location or internal to the fusion protein. In oneembodiment, the fusion protein comprises an affinity ligand (e.g., anenzyme, an antigen, epitope tage) as the first moiety, and a secondmoiety comprising a linker sequence and human CCR2 or a portion thereof.

A “functional fragment or portion”, “functional mutant” and/or“functional fusion protein” of a mammalian CCR2 protein refers to anisolated and/or recombinant protein or polypeptide which has at leastone function characteristic of a mammalian CCR2 protein as describedherein, such as a binding activity, a signaling activity and/or abilityto stimulate a cellular response. Preferred functional variants can binda ligand (i.e., one or more ligands such as MCP-1, MCP-2, MCP-3 and/orMCP-4), and are referred to herein as “ligand binding variants”.

In one embodiment, a functional variant of mammalian CCR2 shares atleast about 85% sequence identity with said mammalian CCR2, preferablyat least about 90% sequence identity, and more preferably at least about95% sequence identity with said mammalian CCR2. The nucleic acid andamino acid sequences of human CCR2a and CCR2b are described in U.S. Pat.No. 5,707,815. Sequence identity can be determine using a suitableprogram, such as the Blastx program (Version 1.4), using appropriateparameters, such as default parameters. In one embodiment, parametersfor Blastx search are scoring matrix BLOSUM62, W=3. In anotherembodiment, a functional variant comprises a nucleic acid sequence whichis different from the naturally-occurring nucleic acid molecule butwhich, due to the degeneracy of the genetic code, encodes mammalian CCR2or a portion thereof.

A composition comprising an isolated and/or recombinant mammalian CCR2or functional variant thereof can be maintained under conditionssuitable for binding, the mammalian CCR2 or variant is contacted with anantibody or fragment to be tested, and binding is detected or measureddirectly or indirectly. In one embodiment, cells which naturally expressCCR2 or cells comprising a recombinant nucleic acid sequence whichencodes a mammalian CCR2 or variant thereof are used. The cells aremaintained under conditions appropriate for expression of receptor. Thecells are contacted with an antibody or fragment under conditionssuitable for binding (e.g., in a suitable binding buffer), and bindingis detected by standard techniques. To determine binding, the extent ofbinding can be determined relative to a suitable control (e.g., comparedwith background determined in the absence of antibody, compared withbinding of a second antibody (i.e., a standard), compared with bindingof antibody to untransfected cells). A cellular fraction, such as amembrane fraction, containing receptor or liposomes comprising receptorcan be used in lieu of whole cells.

In one embodiment, the antibody is labeled with a suitable label (e.g.,fluorescent label, isotope label, antigen or epitope label, enzymelabel), and binding is determined by detection of the label. In anotherembodiment, bound antibody can be detected by labeled second antibody.Specificity of binding can be assessed by competition or displacement,for example, using unlabeled antibody or a ligand as competitor.

Binding inhibition assays can also be used to identify antibodies orfragments thereof which bind CCR2 and inhibit binding of anothercompound such as a ligand (e.g., MCP-1, MCP-2, MCP-3 and/or MCP-4) toCCR2 or a functional variant. For example, a binding assay can beconducted in which a reduction in the binding of a ligand of CCR2 (inthe presence of an antibody), as compared to binding of the ligand inthe absence of the antibody, is detected or measured. A compositioncomprising an isolated and/or recombinant mammalian CCR2 or functionalvariant thereof can be contacted with the ligand and antibodysimultaneously, or one after the other, in either order. A reduction inthe extent of binding of the ligand in the presence of the antibody, isindicative of inhibition of binding by the antibody. For example,binding of the ligand could be decreased or abolished.

In one embodiment, direct inhibition of the binding of a ligand (e.g., achemokine such as MCP-1) to a mammalian CCR2 or variant thereof by anantibody or fragment is monitored. For example, the ability of anantibody to inhibit the binding of ¹²⁵I-labeled MCP-1, ¹²⁵I-labeledMCP-2, ¹²⁵I-labeled MCP-3 or ¹²⁵I-labeled MCP-4 to mammalian CCR2 can bemonitored. Such an assay can be conducted using suitable cells bearingCCR2 or a functional variant thereof, such as isolated blood cells(e.g., T cells, PBMC) or a suitable cell line naturally expressing CCR2,or a cell line containing nucleic acid encoding a mammalian CCR2, or amembrane fraction from said cells, for instance.

Other methods of identifying the presence of an antibody which bindsCCR2 are available, such as other suitable binding assays, or methodswhich monitor events which are triggered by receptor binding, includingsignaling function and/or stimulation of a cellular response (e.g.,leukocyte trafficking).

It will be understood that the inhibitory effect of antibodies of thepresent invention can be assessed in a binding inhibition assay.Competition between antibodies for receptor binding can also be assessedin the method. Antibodies which are identified in this manner can befurther assessed to determine whether, subsequent to binding, they actto inhibit other functions of CCR2 and/or to assess their therapeuticutility.

Signaling Assays

The binding of a ligand or promoter, such as an agonist, to CCR2 canresult in signaling by this G protein-coupled receptor, and the activityof G proteins as well as other intracellular signaling molecules isstimulated. The induction of signaling function by a compound (e.g., anantibody or fragment thereof) can be monitored using any suitablemethod. Such an assay can be used to identify antibody agonists of CCR2.The inhibitory activity of an antibody or functional fragment thereofcan be determined using a ligand or promoter in the assay, and assessingthe ability of the antibody to inhibit the activity induced by ligand orpromoter.

G protein activity, such as hydrolysis of GTP to GDP, or later signalingevents triggered by receptor binding, such as induction of rapid andtransient increase in the concentration of intracellular (cytosolic)free calcium [Ca²⁺]i, can be assayed by methods known in the art orother suitable methods (see e.g., Neote, K. et al., Cell, 72: 415-4251993); Van Riper et al., J. Exp. Med., 177: 851-856 (1993); Dahinden, C.A. et al., J. Exp. Med., 179: 751-756 (1994)).

For example, the functional assay of Sledziewski et al. using hybrid Gprotein coupled receptors can be used to monitor the ability a ligand orpromoter to bind receptor and activate a G protein (Sledziewski et al.,U.S. Pat. No. 5,284,746, the teachings of which are incorporated hereinby reference).

Such assays can be performed in the presence of the antibody or fragmentthereof to be assessed, and the ability of the antibody or fragment toinhibit the activity induced by the ligand or promoter is determinedusing known methods and/or methods described herein.

Chemotaxis and Assays of Cellular Stimulation

Chemotaxis assays can also be used to assess the ability of an antibodyor functional fragment thereof to block binding of a ligand to mammalianCCR2 or functional variant thereof and/or inhibit function associatedwith binding of the ligand to the receptor. These assays are based onthe functional migration of cells in vitro or in vivo induced by acompound. Chemotaxis can be assessed as described in the Examples, e.g.,in an assay utilizing a 96-well chemotaxis plate, or using otherart-recognized methods for assessing chemotaxis. For example, the use ofan in vitro transendothelial chemotaxis assay is described by Springeret al. (Springer et al., WO 94/20142, published Sep. 15, 1994, theteachings of which are incorporated herein by reference; see also Bermanet al., Immunol. Invest. 17: 625-677 (1988)). Migration acrossendothelium into collagen gels has also been described (Kavanaugh etal., J. Immunol., 146: 4149-4156 (1991)). Stable transfectants of mouseL1-2 pre-B cells or of other suitable host cells capable of chemotaxiscan be used in chemotaxis assays, for example.

Generally, chemotaxis assays monitor the directional movement ormigration of a suitable cell (such as a leukocyte (e.g., lymphocyte,eosinophil, basophil)) into or through a barrier (e.g., endothelium, afilter), toward increased levels of a compound, from a first surface ofthe barrier toward an opposite second surface. Membranes or filtersprovide convenient barriers, such that the directional movement ormigration of a suitable cell into or through a filter, toward increasedlevels of a compound, from a first surface of the filter toward anopposite second surface of the filter, is monitored. In some assays, themembrane is coated with a substance to facilitate adhesion, such asICAM-1, fibronectin or collagen. Such assays provide an in vitroapproximation of leukocyte “homing”.

For example, one can detect or measure inhibition of the migration ofcells in a suitable container (a containing means), from a first chamberinto or through a microporous membrane into a second chamber whichcontains an antibody to be tested, and which is divided from the firstchamber by the membrane. A suitable membrane, having a suitable poresize for monitoring specific migration in response to compound,including, for example, nitrocellulose, polycarbonate, is selected. Forexample, pore sizes of about 3-8 microns, and preferably about 5-8microns can be used. Pore size can be uniform on a filter or within arange of suitable pore sizes.

To assess migration and inhibition of migration, the distance ofmigration into the filter, the number of cells crossing the filter thatremain adherent to the second surface of the filter, and/or the numberof cells that accumulate in the second chamber can be determined usingstandard techniques (e.g., microscopy). In one embodiment, the cells arelabeled with a detectable label (e.g., radioisotope, fluorescent label,antigen or epitope label), and migration can be assessed in the presenceand absence of the antibody or fragment by determining the presence ofthe label adherent to the membrane and/or present in the second chamberusing an appropriate method (e.g., by detecting radioactivity,fluorescence, immunoassay). The extent of migration induced by anantibody agonist can be determined relative to a suitable control (e.g.,compared to background migration determined in the absence of theantibody, compared to the extent of migration induced by a secondcompound (i.e., a standard), compared with migration of untransfectedcells induced by the antibody).

In one embodiment, particularly for T cells, monocytes or cellsexpressing a mammalian CCR2, transendothelial migration can bemonitored. In this embodiment, transmigration through an endothelialcell layer is assessed. To prepare the cell layer, endothelial cells canbe cultured on a microporous filter or membrane, optionally coated witha substance such as collagen, fibronectin, or other extracellular matrixproteins, to facilitate the attachment of endothelial cells. Preferably,endothelial cells are cultured until a confluent monolayer is formed. Avariety of mammalian endothelial cells can are available for monolayerformation, including for example, vein, artery or microvascularendothelium, such as human umbilical vein endothelial cells (CloneticsCorp, San Diego, Calif.). To assay chemotaxis in response to aparticular mammalian receptor, endothelial cells of the same mammal arepreferred; however endothelial cells from a heterologous mammalianspecies or genus can also be used.

Generally, the assay is performed by detecting the directional migrationof cells into or through a membrane or filter, in a direction towardincreased levels of a compound, from a first surface of the filtertoward an opposite second surface of the filter, wherein the filtercontains an endothelial cell layer on a first surface. Directionalmigration occurs from the area adjacent to the first surface, into orthrough the membrane, towards a compound situated on the opposite sideof the filter. The concentration of compound present in the areaadjacent to the second surface, is greater than that in the areaadjacent to the first surface.

In one embodiment used to test for an antibody inhibitor, a compositioncomprising cells capable of migration and expressing a mammalian CCR2receptor can be placed in the first chamber. A composition comprisingone or more ligands or promoters capable of inducing chemotaxis of thecells in the first chamber (having chemoattractant function) is placedin the second chamber. Preferably shortly before the cells are placed inthe first chamber, or simultaneously with the cells, a compositioncomprising the antibody to be tested is placed, preferably, in the firstchamber. Antibodies or functional fragments thereof which can bindreceptor and inhibit the induction of chemotaxis, by a ligand orpromoter, of the cells expressing a mammalian CCR2 in this assay areinhibitors of receptor function (e.g., inhibitors of stimulatoryfunction). A reduction in the extent of migration induced by the ligandor promoter in the presence of the antibody or fragment is indicative ofinhibitory activity. Separate binding studies (see above) could beperformed to determine whether inhibition is a result of binding of theantibody to receptor or occurs via a different mechanism.

In vivo assays which monitor leukocyte infiltration of a tissue, inresponse to injection of a compound (e.g., chemokine or antibody) in thetissue, are described below (see Models of Inflammation). These modelsof in vivo homing measure the ability of cells to respond to a ligand orpromoter by emigration and chemotaxis to a site of inflammation and toassess the ability of an antibody or fragment thereof to block thisemigration.

In addition to the methods described, the effects of an antibody orfragment on the stimulatory function of CCR2 can be assessed bymonitoring cellular responses induced by active receptor, using suitablehost cells containing receptor.

Identification of Additional Ligands, Inhibitors and/or Promoters ofMammalian CCR2 Function

The assays described above, which can be used to assess binding andfunction of the antibodies and fragments of the present invention, canbe adapted to identify additional ligands or other substances which binda mammalian CCR2 or functional variant thereof, as well as inhibitorsand/or promoters of mammalian CCR2 function. For example, agents havingthe same or a similar binding specificity as that of an antibody of thepresent invention or functional portion thereof can be identified by acompetition assay with said antibody or portion thereof. Thus, thepresent invention also encompasses methods of identifying ligands of thereceptor or other substances which bind a mammalian CCR2 protein, aswell as inhibitors (e.g., antagonists) or promoters (e.g., agonists) ofreceptor function. In one embodiment, cells bearing a mammalian CCR2protein or functional variant thereof (e.g., leukocytes, cell lines orsuitable host cells which have been engineered to express a mammalianCCR2 protein or functional variant encoded by a nucleic acid introducedinto said cells) are used in an assay to identify and assess theefficacy of ligands or other substances which bind receptor, includinginhibitors or promoters of receptor function. Such cells are also usefulin assessing the function of the expressed receptor protein orpolypeptide.

According to the present invention, ligands and other substances whichbind receptor, inhibitors and promoters of receptor function can beidentified in a suitable assay, and further assessed for therapeuticeffect. Inhibitors of receptor function can be used to inhibit (reduceor prevent) receptor activity, and ligands and/or promoters can be usedto induce (trigger or enhance) normal receptor function where indicated.Thus, the present invention provides a method of treating inflammatorydiseases, including autoimmune disease and graft rejection, comprisingadministering an inhibitor of receptor function to an individual (e.g.,a mammal). The present invention further provides a method ofstimulating receptor function by administering a novel ligand orpromoter of receptor function to an individual, providing a new approachto selective stimulation of leukocyte function, which is useful, forexample, in the treatment of infectious diseases and cancer.

As used herein, a “ligand” of a mammalian CCR2 protein refers to aparticular class of substances which bind to a mammalian CCR2 protein,including natural ligands and synthetic and/or recombinant forms ofnatural ligands. Infectious agents having a tropism for mammalianCCR2-positive cells (e.g., viruses such as HIV) can also bind to amammalian CCR2 protein. A natural ligand of a selected mammalianreceptor is of a mammalian origin which is the same as that of themammalian CCR2 protein (e.g., a chemokine such as MCP-1, MCP-2, MCP-3and/or MCP-4). In a preferred embodiment, ligand binding of a mammalianCCR2 protein occurs with high affinity.

As used herein, an “inhibitor” is a substance which inhibits (decreasesor prevents) at least one function characteristic of a mammalian CCR2protein (e.g., a human CCR2), such as a binding activity (e.g., ligandbinding, promoter binding, antibody binding), a signaling activity(e.g., activation of a mammalian G protein, induction of rapid andtransient increase in the concentration of cytosolic free calcium[Ca²⁺]i), and/or cellular response function (e.g., stimulation ofchemotaxis, exocytosis or inflammatory mediator release by leukocytes).An inhibitor is also a substance which inhibits HIV entry into a cell.The term inhibitor refers to substances including antagonists which bindreceptor (e.g., an antibody, a mutant of a natural ligand, smallmolecular weight organic molecules, other competitive inhibitors ofligand binding), and substances which inhibit receptor function withoutbinding thereto (e.g., an anti-idiotypic antibody).

As used herein, a “promoter” is a substance which promotes (induces,causes, enhances or increases) at least one function characteristic of amammalian CCR2 protein (e.g., a human CCR2), such as a binding activity(e.g., ligand, inhibitor and/or promoter binding), a signaling activity(e.g., activation of a mammalian G protein, induction of rapid andtransient increase in the concentration of cytosolic free calcium[Ca²⁺]i), and/or a cellular response function (e.g., stimulation ofchemotaxis, exocytosis or inflammatory mediator release by leukocytes).The term promoter refers to substances including agonists which bindreceptor (e.g., an antibody, a homolog of a natural ligand from anotherspecies), and substances which promote receptor function without bindingthereto (e.g., by activating an associated protein). In a preferredembodiment, the agonist is other than a homolog of a natural ligand.

Thus, the invention also relates to a method of detecting or identifyingan agent which binds a mammalian CC-chemokine receptor 2 or ligandbinding variant thereof, including ligands, inhibitors, promoters, andother substances which bind a mammalian CCR2 receptor or functionalvariant. According to the method, an agent to be tested, an antibody orantigen-binding fragment of the present invention (e.g., 8G2, 1D9, anantibody having an epitopic specificity which is the same as or similarto that of 8G2 or 1D9, and antigen-binding fragments thereof) and acomposition comprising a mammalian CC-chemokine receptor 2 or a ligandbinding variant thereof can be combined. The foregoing components arecombined under conditions suitable for binding of the antibody orantigen-binding fragment to mammalian CC-chemokine receptor 2 or aligand binding variant thereof, and binding of the antibody or fragmentto the mammalian CC-chemokine receptor 2 or ligand binding variant isdetected or measured, either directly or indirectly, according tomethods described herein or other suitable methods. A decrease in theamount of complex formed relative to a suitable control (e.g., in theabsence of the agent to be tested) is indicative that the agent bindssaid receptor or variant. The composition comprising a mammalianCC-chemokine receptor 2 or a ligand binding variant thereof can be amembrane fraction of a cell bearing recombinant chemokine receptor 2protein or ligand binding variant thereof. The antibody or fragmentthereof can be labeled with a label such as a radioisotope, spin label,antigen or epitope label, enzyme label, fluorescent group andchemiluminescent group.

In one embodiment, the invention relates to a method of detecting oridentifying an agent which binds a mammalian CC-chemokine receptor 2 ora ligand binding variant thereof, comprising combining an agent to betested, an antibody or antigen-binding fragment of the present invention(e.g., 1D9, 8G2, an antibody having an epitopic specificity which is thesame as or similar to that of 1D9 or 8G2, or antigen-binding fragmentsthereof) and a cell bearing a mammalian CC-chemokine receptor 2 or aligand binding variant thereof. The foregoing components are combinedunder conditions suitable for binding of the antibody or antigen-bindingfragment to the CCR2 protein or ligand binding variant thereof, andbinding of the antibody or fragment to the mammalian CC-chemokinereceptor 2 or variant is detected or measured, either directly orindirectly, by methods described herein and or other suitable methods. Adecrease in the amount of complex formed relative to a suitable controlis indicative that the agent binds the receptor or variant. The antibodyor fragment thereof can be labeled with a label selected from the groupconsisting of a radioisotope, spin label, antigen or epitope label,enzyme label, fluorescent group and chemiluminescent group. These andsimilar assays can be used to detect agents, including ligands (e.g.,chemokines or strains of HIV which interact with CCR2) or othersubstances, including inhibitors or promoters of receptor function,which can bind CCR2 and compete with the antibodies described herein forbinding to the receptor.

The assays described above can be used, alone or in combination witheach other or other suitable methods, to identify ligands or othersubstances which bind a mammalian CCR2 protein, and inhibitors orpromoters of a mammalian CCR2 protein or variant. The in vitro methodsof the present invention can be adapted for high-throughput screening inwhich large numbers of samples are processed (e.g., a 96-well format).Cells expressing mammalian CCR2 (e.g., human CCR2) at levels suitablefor high-throughput screening can be used, and thus, are particularlyvaluable in the identification and/or isolation of ligands or othersubstances which bind receptor, and inhibitors or promoters of mammalianCCR2 proteins. Expression of receptor can be monitored in a variety ofways. For instance, expression can be monitored using antibodies of thepresent invention which bind receptor or a portion thereof. Also,commercially available antibodies can be used to detect expression of anantigen- or epitope-tagged fusion protein comprising a receptor proteinor polypeptide (e.g., FLAG tagged receptors), and cells expressing thedesired level can be selected.

Nucleic acid encoding a mammalian CCR2 protein or functional variantthereof can be incorporated into an expression system to produce areceptor protein or polypeptide. An isolated and/or recombinantmammalian CCR2 protein or variant, such as a receptor expressed in cellsstably or transiently transfected with a construct comprising arecombinant nucleic acid encoding a mammalian CCR2 protein or variant,or in a cell fraction containing receptor (e.g., a membrane fractionfrom transfected cells, liposomes incorporating receptor), can be usedin tests for receptor function. The receptor can be further purified ifdesired. Testing of receptor function can be carried out in vitro or invivo.

An isolated and/or recombinant mammalian CCR2 protein or functionalvariant thereof, such as a human CCR2, can be used in the presentmethod, in which the effect of a compound is assessed by monitoringreceptor function as described herein or using other suitabletechniques. For example, stable or transient transfectants (e.g.,baculovirus infected Sf9 cells, stable tranfectants of mouse L1/2 pre-Bcells), can be used in binding assays. Stable transfectants of Jurkatcells or of other suitable cells capable of chemotaxis can be used(e.g., mouse L1/2 pre-B cells) in chemotaxis assays, for example.

According to the method of the present invention, compounds can beindividually screened or one or more compounds can be testedsimultaneously according to the methods herein. Where a mixture ofcompounds is tested, the compounds selected by the processes describedcan be separated (as appropriate) and identified by suitable methods(e.g., PCR, sequencing, chromatography, mass spectroscopy). The presenceof one or more compounds (e.g., a ligand, inhibitor, promoter) in a testsample can also be determined according to these methods.

Large combinatorial libraries of compounds (e.g., organic compounds,recombinant or synthetic peptides, “peptoids”, nucleic acids) producedby combinatorial chemical synthesis or other methods can be tested (seee.g., Zuckerman, R. N. et al., J. Med. Chem., 37: 2678-2685 (1994) andreferences cited therein; see also, Ohlmeyer, M. H. J. et al., Proc.Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H. et al.,Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to taggedcompounds; Rutter, W. J. et al. U.S. Pat. No. 5,010,175; Huebner, V. D.et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No.4,833,092). Where compounds selected from a combinatorial library by thepresent method carry unique tags, identification of individual compoundsby chromatographic methods is possible.

In one embodiment, phage display methodology is used. For example, amammalian CCR2 protein or functional variant, an antibody or functionalportion thereof of the present invention, and a phage (e.g., a phage orcollection of phage such as a library) displaying a polypeptide, can becombined under conditions appropriate for binding of the antibody orportion thereof to the mammalian CCR2 protein or variant (e.g., in asuitable binding buffer). Phage which can compete with the antibody orportion thereof and bind to the mammalian CCR2 protein or variant can bedetected or selected using standard techniques or other suitablemethods. Bound phage can be separated from receptor using a suitableelution buffer. For example, a change in the ionic strength or pH canlead to a release of phage. Alternatively, the elution buffer cancomprise a release component or components designed to disrupt bindingof compounds (e.g., one or more compounds which can disrupt binding ofthe displayed peptide to the receptor, such as a ligand, inhibitor,and/or promoter which competitively inhibits binding). Optionally, theselection process can be repeated or another selection step can be usedto further enrich for phage which bind receptor. The displayedpolypeptide can be characterized (e.g., by sequencing phage DNA). Thepolypeptides identified can be produced and further tested for binding,and for inhibitor or promoter function. Analogs of such peptides can beproduced which will have increased stability or other desirableproperties.

In one embodiment, phage expressing and displaying fusion proteinscomprising a coat protein with an N-terminal peptide encoded by randomsequence nucleic acids can be produced. Suitable host cells expressing amammalian CCR2 protein or variant and an anti-CCR2 antibody orfunctional portion thereof, are combined with the phage, bound phage areselected, recovered and characterized. (See e.g., Doorbar, J. and G.Winter, J. Mol. Biol., 244: 361 (1994) discussing a phage displayprocedure used with a G protein-coupled receptor).

Other sources of potential ligands or other substances which bind to, orinhibitors and/or promoters of, mammalian CCR2 proteins include, but arenot limited to, variants of CCR2 ligands, including naturally occurring,synthetic or recombinant variants of MCP-1, MCP-2, MCP-3 and/or MCP-4,substances such as other chemoattractants or chemokines, variantsthereof, low molecular weight organic molecules, other inhibitors and/orpromoters (e.g., anti-CCR2 antibodies, antagonists, agonists), other Gprotein-coupled receptor ligands, inhibitors and/or promoters (e.g.,antagonists or agonists), and soluble portions of a mammalian CCR2receptor, such as a suitable receptor peptide or analog which caninhibit receptor function (see e.g., Murphy, R. B., WO 94/05695).

Models of Inflammation

In vivo models of inflammation are available which can be used to assessthe effects of antibodies and fragments of the invention in vivo astherapeutic agents. For example, leukocyte infiltration upon intradermalinjection of a chemokine and an antibody or fragment thereof reactivewith mammalian CCR2 into a suitable animal, such as rabbit, mouse, rat,guinea pig or rhesus macaque can be monitored (see e.g., Van Damme, J.et al., J. Exp. Med., 176: 59-65 (1992); Zachariae, C. O. C. et al., J.Exp. Med. 171: 2177-2182 (1990); Jose, P. J. et al., J. Exp. Med. 179:881-887 (1994)). In one embodiment, skin biopsies are assessedhistologically for infiltration of leukocytes (e.g., eosinophils,granulocytes). In another embodiment, labeled cells (e.g., stablytransfected cells expressing a mammalian CCR2, labeled with ¹¹¹In forexample) capable of chemotaxis and extravasation are administered to theanimal. For example, an antibody or fragment to be assessed can beadministered, either before, simultaneously with or after ligand oragonist is administered to the test animal. A decrease of the extent ofinfiltration in the presence of antibody as compared with the extent ofinfiltration in the absence of inhibitor is indicative of inhibition.

Diagnostic and Therapeutic Applications

The antibodies and fragments of the present invention are useful in avariety of applications, including research, diagnostic and therapeuticapplications. In one embodiment, the antibodies are labeled with asuitable label (e.g., fluorescent label, chemiluminescent label, isotopelabel, antigen or epitope label or enzyme label). For instance, they canbe used to isolate and/or purify receptor or portions thereof, and tostudy receptor structure (e.g., conformation) and function.

In addition, the various antibodies of the present invention can be usedto detect CCR2 or to measure the expression of receptor, for example, onT cells (e.g., CD8+ cells, CD45RO+ cells), monocytes and/or on cellstransfected with a receptor gene. Thus, they also have utility inapplications such as cell sorting (e.g., flow cytometry, fluorescenceactivated cell sorting), for diagnostic or research purposes.

The anti-CCR2 antibodies of the present invention have value indiagnostic applications. An anti-CCR2 antibody or fragment thereof canbe used to monitor expression of this receptor in HIV infectedindividuals, similar to the way anti-CD4 has been used as a diagnosticindicator of disease stage.

Typically, diagnostic assays entail detecting the formation of a complexresulting from the binding of an antibody or fragment thereof to CCR2.For diagnostic purposes, the antibodies or antigen-binding fragments canbe labeled or unlabeled. The antibodies or fragments can be directlylabeled. A variety of labels can be employed, including, but not limitedto, radionuclides, fluorescers, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654 and4,098,876). When unlabeled, the antibodies or fragments can be detectedusing suitable means, as in agglutination assays, for example. Unlabeledantibodies or fragments can also be used in combination with another(i.e., one or more) suitable reagent which can be used to detectantibody, such as a labeled antibody (e.g., a second antibody) reactivewith the first antibody (e.g., anti-idiotype antibodies or otherantibodies that are specific for the unlabeled immunoglobulin) or othersuitable reagent (e.g., labeled protein A).

In one embodiment, the antibodies or fragments of the present inventioncan be utilized in enzyme immunoassays, wherein the subject antibody orfragment, or second antibodies, are conjugated to an enzyme. When abiological sample comprising a mammalian CCR2 protein is combined withthe subject antibodies, binding occurs between the antibodies and CCR2protein. In one embodiment, a sample containing cells expressing amammalian CCR2 protein, such as human blood, is combined with thesubject antibodies, and binding occurs between the antibodies and cellsbearing a human CCR2 protein comprising an epitope recognized by theantibody. These bound cells can be separated from unbound reagents andthe presence of the antibody-enzyme conjugate specifically bound to thecells can be determined, for example, by contacting the sample with asubstrate of the enzyme which produces a color or other detectablechange when acted on by the enzyme. In another embodiment, the subjectantibodies can be unlabeled, and a second, labeled antibody can be addedwhich recognizes the subject antibody.

Kits for use in detecting the presence of a mammalian CCR2 protein in abiological sample can also be prepared. Such kits will include anantibody or functional fragment thereof which binds to a mammalianCC-chemokine receptor 2 or portion of said receptor, as well as one ormore ancillary reagents suitable for detecting the presence of a complexbetween the antibody or fragment and CCR2 or portion thereof. Theantibody compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The antibodies, which can belabeled or unlabeled, can be included in the kits with adjunctingredients (e.g., buffers, such as Tris, phosphate and carbonate,stabilizers, excipients, biocides and/or inert proteins, e.g., bovineserum albumin). For example, the antibodies can be provided as alyophilized mixture with the adjunct ingredients, or the adjunctingredients can be separately provided for combination by the user.Generally these adjunct materials will be present in less than about 5%weight based on the amount of active antibody, and usually will bepresent in a total amount of at least about 0.001% weight based onantibody concentration. Where a second antibody capable of binding tothe monoclonal antibody is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

Similarly, the present invention also relates to a method of detectingand/or quantitating expression of a mammalian CCR2 or a portion of thereceptor by a cell, in which a composition comprising a cell or fractionthereof (e.g., membrane fraction) is contacted with an antibody orfunctional fragment thereof (e.g., 1D9 and/or 8G2) which binds to amammalian CCR2 or portion of the receptor under conditions appropriatefor binding of the antibody or fragment thereto, and binding ismonitored. Detection of the antibody, indicative of the formation of acomplex between antibody and CCR2 or a portion thereof, indicates thepresence of the receptor. Binding of antibody to the cell can bedetermined as described above under the heading “Binding Assays”, forexample. The method can be used to detect expression of CCR2 on cellsfrom an individual (e.g., in a sample, such as a body fluid, such asblood, saliva or other suitable sample). The level of expression of CCR2on the surface of T cells or monocytes can also be determined, forinstance, by flow cytometry, and the level of expression (e.g., stainingintensity) can be correlated with disease susceptibility, progression orrisk.

Chemokine receptors function in the migration of leukocytes throughoutthe body, particularly to inflammatory sites. Inflammatory cellemigration from the vasculature is regulated by a three-step processinvolving interactions of leukocyte and endothelial cell adhesionproteins and cell specific chemoattractants and activating factors(Springer, T. A., Cell, 76:301-314 (1994); Butcher, E. C., Cell,67:1033-1036 (1991); Butcher, E. C. and Picker, L. J., Science (Wash.DC), 272:60-66 (1996)). These are: (a) a low affinity interactionbetween leukocyte selectins and endothelial cell carbohydrates; (b) ahigh-affinity interaction between leukocyte chemoattractant receptorsand chemoattractant/activating factors; and (c) a tight-binding betweenleukocyte integrins and endothelial cell adhesion proteins of theimmunoglobulin superfamily. Different leukocyte subsets expressdifferent repertoires of selectins, chemoattractant receptors andintegrins. Additionally, inflammation alters the expression ofendothelial adhesion proteins and the expression of chemoattractant andleukocyte activating factors. As a consequence, there is a great deal ofdiversity for regulating the selectivity of leukocyte recruitment toextravascular sites. The second step is crucial in that the activationof the leukocyte chemoattractant receptors is thought to cause thetransition from the selectin-mediated cell rolling to theintegrin-mediated tight binding. This results in the leukocyte beingready to transmigrate to perivascular sites. Thechemoattractant/chemoattractant receptor interaction is also crucial fortransendothelial migration and localization within a tissue (Campbell,J. J., et al., J. Cell Biol., 134:255-266 (1996); Carr, M. W., et al.,Immunity, 4:179-187 (1996)). This migration is directed by aconcentration gradient of chemoattractant leading towards theinflammatory focus.

CCR2 has an important role in leukocyte trafficking. It is likely thatCCR2 is a key chemokine receptor for T cell or T cell subset or monocytemigration to certain inflammatory sites, and so anti-CCR2 mAbs can beused to inhibit (reduce or prevent) T cell or monocyte migration,particularly that associated with T cell dysfunction, such as autoimmunedisease, or allergic reactions or with monocyte-mediated disorders suchas atherosclerosis. Accordingly, the antibodies and fragments thereof ofthe present invention can also be used to modulate receptor function inresearch and therapeutic applications. For instance, the antibodies andfunctional fragments described herein can act as inhibitors to inhibit(reduce or prevent) (a) binding (e.g., of a ligand, an inhibitor or apromoter) to the receptor, (b) a receptor signaling function, and/or (c)a stimulatory function. Antibodies which act as inhibitors of receptorfunction can block ligand or promoter binding directly or indirectly(e.g., by causing a conformational change). For example, antibodies caninhibit receptor function by inhibiting binding of a ligand, or bydesensitization (with or without inhibition of binding of a ligand).Antibodies which bind receptor can also act as agonists of receptorfunction, triggering or stimulating a receptor function, such as asignaling and/or a stimulatory function of a receptor (e.g., leukocytetrafficking) upon binding to receptor.

Thus, the present invention provides a method of inhibiting leukocytetrafficking in a mammal (e.g., a human patient), comprisingadministering to the mammal an effective amount of an antibody orfunctional fragment of the present invention. Administration of anantibody or fragment of the present invention can result in ameliorationor elimination of the disease state.

The antibody of the present invention, or a functional fragment thereof,can also be used to treat disorders in which activation of the CCR2receptor by binding of chemokines is implicated. For example, theantibodies or functional fragments thereof (e.g., 1D9 and/or 8G2 orfunctional fragments thereof) can be used to treat allergy,atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation,histamine and IgE-mediated allergic reactions, shock, and rheumatoidarthritis, atherosclerosis, multiple sclerosis, stenosis, restenosis,allograft rejection, fibrotic disease, asthma, and inflammatoryglomerulopathies.

Diseases or conditions of humans or other species which can be treatedwith inhibitors of CCR2 receptor function (including antibodies orsuitable fragments thereof), include, but are not limited to:

-   -   inflammatory or allergic diseases and conditions, including        respiratory allergic diseases such as asthma, allergic rhinitis,        hypersensitivity lung diseases, hypersensitivity pneumonitis,        interstitial lung diseases (ILD) (e.g., idiopathic pulmonary        fibrosis, or ILD associated with rheumatoid arthritis, systemic        lupus erythematosus, ankylosing spondylitis, systemic sclerosis,        Sjogren's syndrome, polymyositis or dermatomyositis);        anaphylaxis or hypersensitivity responses, drug allergies (e.g.,        to penicillin, cephalosporins), insect sting allergies;        inflammatory bowel diseases, such as Crohn's disease and        ulcerative colitis; spondyloarthropathies; scleroderma;        psoriasis and inflammatory dermatoses such as dermatitis,        eczema, atopic dermatitis, allergic contact dermatitis,        urticaria; vasculitis (e.g., necrotizing, cutaneous, and        hypersensitivity vasculitis);    -   autoimmune diseases, such as arthritis (e.g., rheumatoid        arthritis, psoriatic arthritis), multiple sclerosis, systemic        lupus erythematosus, myasthenia gravis, juvenile onset diabetes,        nephritides such as glomerulonephritis, autoimmune thyroiditis,        Behcet's disease;    -   graft rejection (e.g., in transplantation), including allograft        rejection or graft-versus-host disease, and organ        transplant-associated arteriosclerosis;    -   atherosclerosis;    -   cancers with leukocyte infiltration of the skin or organs;    -   stenosis or restenosis of the vasculature, particularly of the        arteries, e.g., the coronary artery, such as stenosis or        restenosis which results from vascular intervention (e.g.,        surgical, therapeutic or mechanical intervention), as well as        neointimal hyperplasia. For example, restenosis, which typically        produces a narrowing of the lumenal opening of the vessel, can        result from vascular injury including, but not limited to, that        produced by vascular graft procedures, angioplasty, including        angioplasty performed by balloon, atherectomy, laser or other        suitable method (e.g., percutaneous translumenal coronary        angioplasty (PTCA)), stent placement (e.g., mechanical or        biological endovascular stent placement), vascular bypass        procedures or combinations thereof, as well as other procedures        used to treat stenotic or occluded blood vessels;    -   other diseases or conditions (including CCR2-mediated diseases        or conditions), in which undesirable inflammatory responses are        to be inhibited can be treated, including, but not limited to,        reperfusion injury, certain hematologic malignancies,        cytokine-induced toxicity (e.g., septic shock, endotoxic shock),        polymyositis, dermatomyositis, and granulomatous diseases        including sarcoidosis.

Diseases or conditions of humans or other species which can be treatedwith promoters of CCR2 receptor function (including antibodies orfragments thereof), include, but are not limited to:

-   -   immunosuppression, such as that in individuals with        immunodeficiency syndromes such as AIDS, individuals undergoing        radiation therapy, chemotherapy, therapy for autoimmune disease        or other drug therapy (e.g., corticosteroid therapy), which        causes immunosuppression; and immunosuppression due congenital        deficiency in receptor function or other causes.

Anti-CCR2 antibodies of the present invention can block the binding ofone or more chemokines, thereby blocking the downstream cascade of oneor more events leading to the above disorders.

Antibodies and functional fragments thereof which are antagonists ofCCR2 can be used as therapeutics for AIDS, as well as certaininflammatory diseases. HIV-1 and HIV-2 are the etiologic agents ofacquired immunodeficiency syndrome (AIDS) in humans. AIDS results inpart from the depletion of CD4+ T lymphocytes in HIV infectedindividuals. HIV-1 infects primarily T lymphocytes,monocytes/macrophages, dendritic cells and, in the central nervoussystem, microglia. All of these cells express the CD4 glycoprotein,which serves as a receptor for HIV-1 and HIV-2. Efficient entry of HIVinto target cells is dependent upon binding of the viral exteriorenvelope glycoprotein, gp120, to the amino-terminal CD4 domain. Aftervirus binding, the HIV-1 envelope glycoproteins mediate the fusion ofviral and host cell membranes to complete the entry process. Membranefusion directed by HIV-1 envelope glycoproteins expressed on theinfected cell surface leads to cell-cell fusion, resulting in syncytia.

Recently, host cell factors in addition to CD4 have been suggested todetermine the efficiency of HIV-1 envelope glycoprotein-mediatedmembrane fusion. The 7 transmembrane receptor (7TMR) termed HUMSTSR,LESTR, or “fusin” has been shown to allow a range of CD4-expressingcells to support infection and cell fusion mediated bylaboratory-adapted HIV-1 envelope glycoproteins (Feng, Y., et al.,Science (Wash. DC), 272:872-877 (1996)). Antibodies to HUMSTSR blockedcell fusion and infection by laboratory-adapted HIV-1 isolates but notby macrophage-tropic primary viruses in vitro (Feng, Y., et al., Science(Wash. DC), 272:872-877 (1996)).

The ability of chemokine receptors and related molecules to facilitatethe infection of primary clinical HIV-1 isolates has been reportedrecently by several groups (see e.g., Bates, P., Cell, 86:1-3 (1996);Choe, H., et al., Cell, 85:1135-1148 (1996); Doranz et al., Cell85:1149-1158 (1996)). These studies indicated that involvement ofvarious members of the chemokine receptor family in the early stages ofHIV-1 infection helps to explain viral tropism and β-chemokineinhibition of primary HIV-1 isolates.

The present invention also provides a method of inhibiting HIV infectionof a cell (e.g., new infection and/or syncytium formation) whichexpresses a mammalian CCR2 or portion thereof, comprising contacting thecell with a composition comprising an effective amount of an antibody orfunctional fragment thereof which binds to a mammalian CCR2 or portionof said receptor. The composition can also comprise one or moreadditional agents effective against HIV, including, but not limited to,anti-CCR3 antibodies, anti-CCR5 antibodies, and anti-fusin antibodies.

Various methods can be used to assess binding of HIV to a cell and/orinfection of a cell by HIV in the presence of the antibodies of thepresent invention. For example, assays which assess binding of gp120 ora portion thereof to the receptor, HIV infection and syncytium formationcan be used (see, for example, Choe, H., et al., Cell, 85:1135-1148(1996)). The ability of the antibody of the present invention to inhibitthese processes can be assessed using these or other suitable methods.

In addition, the present invention provides a method of treating HIV ina patient, comprising administering to the patient a compositioncomprising an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 or portion of said receptor.Again, the composition can also comprise one or more additional agentseffective against HIV, including, but not limited to, anti-CCR3antibodies, anti-CCR5 antibodies, and anti-fusin antibodies. Therapeuticuse of antibody to treat HIV includes prophylactic use (e.g., fortreatment of a patient who may be or who may have been exposed to HIV).For example, health care providers who may be exposed or who have beenexposed to HIV (e.g., by needle-stick) can be treated according to themethod. Another example is the treatment of a patient exposed to virusafter unprotected sexual contact or failure of protection.

In AIDS, multiple drug treatment appears the most promising. Ananti-chemokine receptor antagonist that inhibits HIV infection can beadded to the drug treatment regimen, in particular by blocking virusinfection of new cells. Thus, administration of an antibody or fragmentof the present invention in combination with one or more othertherapeutic agents such as nucleoside analogues (e.g., AZT, 3TC, ddI)and/or protease inhibitors is envisioned, and provides an importantaddition to an HIV treatment regimen. In one embodiment, a humanizedanti-CCR2 mAb is used in combination with a (i.e., one or more)therapeutic agent to reduce viral load from patients, by preventingfusion and/or infection of new cells. Such an antibody can also beuseful in preventing perinatal infection.

Another aspect of the invention relates to a method of preventing HIVinfection in an individual, comprising administering to the individualan effective amount of an antibody or functional fragment thereof whichbinds to CCR2. According to the method, preventing HIV infectionincludes treatment in order to prevent (reduce or eliminate) infectionof new cells in an infected individual or in order to prevent infectionin an individual who may be, may have been, or has been, exposed to HIV.For example, individuals such as an HIV infected individual, a fetus ofan HIV infected female, or a health care worker may be treated accordingto the method of the present invention.

Modes of Administration

One or more antibodies or fragments of the present invention can beadministered to an individual by an appropriate route, either alone orin combination with (before, simultaneous with, or after) another drugor agent, or before, simultaneous with or after surgical, mechanical ortherapeutic intervention. For example, the antibodies of the presentinvention can also be used in combination with other monoclonal orpolyclonal antibodies (e.g., in combination with antibodies which bindother chemokine receptors, including, but not limited to, CCR3 and CCR5)or with existing blood plasma products, such as commercially availablegamma globulin and immune globulin products used in prophylactic ortherapeutic treatments. The antibodies or fragments of the presentinvention can be used as separately administered compositions given inconjunction with antibiotics and/or antimicrobial agents.

An effective amount of an antibody or fragment (i.e., one or moreantibodies or fragments) is administered. An effective amount is anamount sufficient to achieve the desired therapeutic (includingprophylactic) effect, under the conditions of administration, such as anamount sufficient for inhibition of a CCR2 function, and thereby,inhibition of an inflammatory response or HIV infection, or an amountsufficient for promotion of a CCR2 function, as indicated.

A variety of routes of administration are possible including, but notnecessarily limited to, oral, dietary, topical, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection orinfusion), inhalation (e.g., intrabronchial, intraocular, intranasal ororal inhalation, intranasal drops), depending on the disease orcondition to be treated. Other suitable methods of administration canalso include rechargeable or biodegradable devices and slow releasepolymeric devices. The pharmaceutical compositions of this invention canalso be administered as part of a combinatorial therapy with otheragents.

Formulation of an antibody or fragment to be administered will varyaccording to the route of administration and formulation (e.g.,solution, emulsion, capsule) selected. An appropriate pharmaceuticalcomposition comprising an antibody or functional fragment thereof to beadministered can be prepared in a physiologically acceptable vehicle orcarrier. A mixture of antibodies and/or fragments can also be used. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. A variety of appropriate aqueouscarriers are known to the skilled artisan, including water, bufferedwater, buffered saline, polyols (e.g., glycerol, propylene glycol,liquid polyethylene glycol), dextrose solution and glycine. Intravenousvehicles can include various additives, preservatives, or fluid,nutrient or electrolyte replenishers (See, generally, Remington'sPharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositionscan optionally contain pharmaceutically acceptable auxiliary substancesas required to approximate physiological conditions such as pH adjustingand buffering agents and toxicity adjusting agents, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride andsodium lactate. The antibodies and fragments of this invention can belyophilized for storage and reconstituted in a suitable carrier prior touse according to art-known lyophilization and reconstitution techniques.The optimum concentration of the active ingredient(s) in the chosenmedium can be determined empirically, according to procedures well knownto the skilled artisan, and will depend on the ultimate pharmaceuticalformulation desired. For inhalation, the antibody or fragment can besolubilized and loaded into a suitable dispenser for administration(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way. The teachings of allreferences cited herein are incorporated herein by reference.

EXAMPLES Example 1 Materials

The following materials were obtained from the indicated sources:

PE-conjugated anti-CD16, PE-conjugated streptavidin, and biotinylatedanti-human IgE were from Pharmingen (San Diego, Calif.). FITC-conjugatedgoat anti-mouse IgG was from Jackson Immunoresearch Laboratories (WestGrove, Pa.).

FACS Lysing Buffer was from Becton Dickenson (Mountain View, Calif.) and¹²⁵I MCP-1 was from NEN (Boston, Mass.).

Cells, Cell Lines, and Tissue Culture

The murine pre-B lymphoma cell line L1/2 was maintained in RPMI-1640supplemented with 10% Fetal Clone I (Gibco BRL, Gaithersburg, Md.) 50Units/mL penicillin (Gibco BRL), 50 μg/mL streptomycin (Gibco BRL), 2 mML-Glutamine (Gibco BRL), and 55 μM β-mercaptoethanol (Gibco BRL). Othercell lines included transfectants of L1/2 cells expressing either CCR1(Campbell, J. et al. (1996) J. Cell Bio., 134:255-266), CCR5 (Wu et al.,Nature 384:179-183 (1996)) grown in the above culture mediumsupplemented with 800 μg/ml active G418. THP-1 cells (ATCC No. TIB202)were grown in accordance with ATCC instructions. PBMC were purified fromheparinized blood as described in Ponath et al., J. Clin. Invest.,97:604-612 (1996).

Preparation of CCR2b Expression Construct and Stable Transfectants

The coding region for the human CCR2b (Charo et al. (1994) Proc. Natl.Acad. Sci. USA, 91:2752) was obtained by RT-PCR amplification asdescribed (Qin, S. et al. (1996) Eur. J. Immunol., 26:640-647). cDNA wasmade using oligo(dT)-priming, and amplification of the CCR2b codingregion was achieved by nested PCR with the following sets of primerswhich correspond to the positions of the CCR2b sequence (GenBankAccession No. U03905; Charo et al., Proc. Natl. Acad. Sci. USA91:2752-2756 (1994)) as indicated:

1) 5′ primer: 5′-TGAGACAAGCCACAAGCTGAAC-3′ (nucleotides 11 to 32; SEQ IDNO: 1); 3′ Primer: 5′-TCTGTATTAGTACACACAGCCC-3′ (nucleotides 1301 to1280; SEQ ID NO: 2); 2) 5′ Primer: 5′-ATGCTGTCCACATCTCGTTCTCGG-3′(nucleotides 81 to 104; SEQ ID NO: 3); 3′ Primer:5′-TTATAAACCAGCCGAGACTTCCTGCTC-3′ (nucleotides 1164 to 1137; SEQ ID NO:4).

The CCR2B cDNA coding region was modified to contain the CD5 signalpeptide leader sequence (Aruffo et al., Cell 61:1303-1313 (1990)). Thepredicted amino acid sequence of this peptide is:

(SEQ ID NO:5) NH₂-Met-Pro-Met-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Thr-Leu-Tyr-Leu-Leu-Gly-Met-Leu-Val-Ala-Ser-Val-Leu- Ala . . .Using PCR with the CCR2b cDNA as template and two overlapping 5′ primersthat contain a BamHI restriction site, encode the CD5 signal peptidesequence and the amino terminal sequence of CCR2b, and a 3′ primerlocated internally in the CCR2b coding region.

5′ CD5 Seq1 primer (SEQ ID NO: 6)5′-GGGGATCCAGAAACCATGCCCATGGGGTCTCTGCAACCGCTGGCCAC CTTGTACCTGCTG-3′5′ CD5 Seq2 primer (SEQ ID NO: 7)5′-GCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCGTGCTAGCGAT GCTGTCCACATCTCGTTC-3′3′ CCR2AB2 primer (SEQ ID NO: 8; U03905 nucleotides 272 to 255)5′-GACGACCAGCATGTTGCC-3′

The 278 base pair amplified fragment was digested with BamHI and Apaland the resulting 209 base pair fragment was inserted at the Apal siteat position 206 of the CCR2b cDNA (GenBank Accession No. U03905) toreplace the endogenous 5′ base pair fragment of CCR2. The resultingsequence that encodes a CCR2b with the CD5 signal peptide leadersequence immediately preceding the receptor initiator methionine wasinserted into the BamHI and Xhol sites of pcDNA3 (Invitrogen, San Diego,Calif.) to create the mammalian expression plasmid pCD5MCPRB. TheCD5-CCR2b fragment was subcloned into the BamH I-Not I site of pCDEF3(Goldman et al., (1996) Biotechniques 21:1013-1015), and this constructwas designated CCR2bDEF3. In this expression vector, the expression ofthe inserted gene is driven by the EF-1α promoter.

Fifty milliliters of L1/2 cells were seeded at 4×10⁵ cells/mL the daybefore the electroporation. On the day of the electroporation, thecells, which had grown up to a density of 1×10⁶/mL, were centrifuged outof their medium and resuspended in 800 μl room temperatureelectroporation buffer (Zajac et al., DNA 7:509-513). 120 mM L-GlutamicAcid (Sigma), 7 mM Mg Acetate (EM Science), 4.3 mM Glucose (Sigma), 17mM K Pipes, pH 6.9 (Sigma), 1 mM EGTA (Sigma), 5 mM ATP, pH 7.0 (Sigma).Twenty-five micrograms Sca I linearized, phenol/chloroform/isoamylalcohol extracted and isopropanol precipitated CCR2bDEF3 plasmid DNA wasplaced in an 0.4 cm gap electroporation curvette. The resuspended cellswere added to the curvette, and a single pulse applied at 450 volts, 960μFd. The cells were then transferred from the curvette to a T-75 flaskcontaining 15 mL L1/2 growth medium (described above, and grown forthree days, at which time the cells were centrifuged out of their mediumand resuspended in L1/2 growth medium additionally supplemented with 1mM sodium pyruvate (Gibco BRL) and 0.8 mg/mL active G418 (Gibco BRL).

Selection of Cells Expressing CCR2b by Chemotaxis

The transfected cells were allowed to grow for eleven days, at whichpoint they were split 1:20 into fresh growth medium. On the sixteenthday, the cells were selected by chemotaxis. 600 μL 1 nM MCP-1 in RPMI1640 supplemented with 0.5% BSA (RPMI/BSA) was placed in the lowerchamber and 1×10⁶ CCR2bDEF3 cells in 100 μl of RPMI/BSA were placed inthe upper chamber of a 3.0 micron pore 24-well chemotaxis plate (BectonDickinson). The cells were allowed to chemotax for four hours and twentyminutes in a 37° C., 5% CO₂, humidified incubator, at which time theupper chamber was removed. This incubation time was chosen at the timeof the experiment because it was sufficiently long for cells respondingto the MCP-1 to chemotax, but short enough to keep the background low.

Secondary Selection of CCR2b Expressing-Cells by FACS Sorting

The cells which had chemotaxed through the membrane and into the lowerchamber were grown up, and further purified by sterile FACS sorting. Tenmillion CCR2bDEF3 cells were centrifuged out of their medium,resuspended in 2.5 mL PBS(+Ca, Mg) supplemented with 1% heat-inactivatedFetal Calf Serum (“HI FCS”) (Gibco BRL) and 2.5 mL sterile filteredanti-CCR2b amino-terminal peptide antibody supernatant 5A11. The cellsand the antibody were mixed and allowed to incubate on ice for thirtyminutes. The cells were then washed twice with PBS (+) (Gibco BRL), andresuspended in 5 mL of a sterile filtered, 1:250 dilution ofFITC-conjugated, affinity-purified F(ab¹)₂ goat anti-mouse IgG (JacksonImmunoResearch Laboratories) in PBS (+) supplemented with 1% HI FCS. Thecells were incubated for thirty minutes on ice in the dark, and thenwashed twice with PBS(+) (GIBCO BRL). The cells were sorted on theFACSCalibur® and the brightest 4% of cells were collected. (FL1≧3×10²).

The sorted cells were allowed to grow up, and they were resorted usingthe same protocol as above. The brightest 1% of cells were collected.(FL1≧3×10³).

Monoclonal Antibody Production

To produce mAbs to CCR2b, transfectants were continually monitored toensure that levels of expression did not drift downward. FACS stainingwas performed periodically to ascertain receptor expression on thetransfectants using the anti CCR2b antibody supernatant 5A11 with goatanti-mouse IgG FITC as the secondary antibody.

Twenty million CCR2bDEF3.L1/2 cells were washed in RPMI 1640 (Gibco BRL)and incubated in RPMI 1640 plus 0.2 mg/mL Mitomycin C for 30 minutes at37° C. The cells were then washed twice with PBS (+) and 2×10⁷ cells in0.5 mL PBS (+) were injected intraperitoneally into a C57 BL/6 femalemouse. This was repeated two more times at two week intervals. Thefourth time, 2×10⁷ cells were resuspended in 0.25 mL and injectedintravenously. Three days after the intravenous injection, the mouse wassacrificed and the spleen removed and the cells fused with the SP2/0cell line as described (Current Protocols in Immunology, John Wiley andSons, New York, 1992).

This set of mice had previously been immunized many times with 2different cell lines as well as a synthetic peptide, but no antibodiesthat stained CCR2 positive cells were generated from several fusions.The above four immunizations with the CCR2bDEF3.L1/2 cell lineexpressing high levels of CCR2b were critical to obtain the describedantibody.

Selecting Single Cell Clone of CCR2 Transfectants by Limiting Dilution

After the mouse received the last injection, the twice sorted cells wereallowed to grow up again, and then they were further purified bylimiting dilution. The cells were plated at 1 and 0.5 cell per well in96 well plates. Subcloned cells from the 0.5 cell per well dilution weregrown up and tested for CCR2b expression by indirect immunofluorescentFACS analysis using the anti-CCR2b antibody supernatant 5A11 with goatanti-mouse IgG FITC as the secondary antibody. The procedure was thesame as described above, except that the staining volume was 100 μl.Four positives were selected and frozen down.

Identification of Positive Monoclonal Antibodies

Immunofluorescent staining analysis using a FACScan® (Becton Dickinson &Co., Mountain View, Calif.) was used to identify the monoclonalantibodies which were reactive with the CCR2b receptor. Hybridomaculture supernatants were assayed in a 96-well format using goatanti-mouse IgG FITC as the secondary antibody. CCR2bDEF3.L1/2 cells wereused to identify monoclonal antibodies reactive with CCR2b, anduntransfected L1/2 cells were used to eliminate monoclonal antibodiesreactive with other cell surface proteins.

FACS Staining—Cultured Cells

For the staining of cultured transfectant cell lines 0.5×10⁶ cells in 50μl were resuspended in PBS+1% FCS in a 96 well polystyrene V-bottomplate. 50 μl of primary antibody supernatants or HT medium (negativecontrol) were added, and the samples were incubated at 4° C. for 30 min.100 μl of PBS were added and the cells were pelleted by centrifugationand washed once with PBS. The pellet was resuspended in 100 μl PBS+1%FCS containing FITC-conjugated goat anti-mouse IgG antibody (a 1:250dilution) and incubated for thirty minutes at 4° C. in the dark. Thecells were washed twice with PBS, resuspended in PBS, and analyzed byflow cytrometry with a FacScan cytometer using the CellQuest software(Becton-Dickenson) Cells were fixed with PBS/1% formaldehyde if theywere not to be analyzed the same day. Monoclonal antibodies 1D9 and 8G2stain CCR2 transfectants but not CCR1 or CCR5 transfectants (FIG. 1).

FACS Staining—Whole Blood

100 μl whole blood was mixed with 100 μL of 1D9 antibody hybridomasupernatants or HT medium (negative control) and incubated at 4° C. for30 min. After one wash with PBS, 100 μL FITC-conjugated goat anti-mouseIgG antibody (a 1:250 dilution) was added to each sample and incubatedfor 30 min. at 4° C. in the dark. Samples were then washed once with PBSif a second color staining is to be done, otherwise washed twice more inPBS. For two color staining 5111 of mouse serum was added to the cellpellets after the single wash, mixed, and incubated for five minutes at4° C. in the dark. Second primary antibodies (or PBS as a negativecontrol) were added (10 μl anti-CD16, 100 μl 1:200 dilution of anti-IgE)and incubated for thirty minutes at 4° C. in the dark. Samples were thenwashed one time with PBS and resuspended in 100 μL streptavidin PE(1:200 PBS+1% BSA) and incubated for fifteen minutes at 4° C. in thedark. Eyrythrocytes were lysed by adding 2 ml of FACS Lysing Buffer toeach sample and incubating at room temperature in the dark for fifteenminutes or until samples were clear. The cells were pelleted bycentrifugation and all but 200 μl of the supernatant was aspirated. Thesamples were analyzed by flow cytometry on a FacScan cytometer using theCellQuest software (Becton-Dickenson). CCR2b is expressed on mostmonocytes, a subpopulation of lymphocytes and a subset of granulocytes(FIGS. 2A-2L). CCR2b is expressed on an IgE-positive population inperipheral blood (basophils) (FIGS. 3A-3I).

MCP-1 Binding Assays

MCP-1 binding was performed in a final volume of 0.1 ml of 50 mM HepespH 7.4, 1 mM CaCl₂, 5 mM MgCl₂, 0.02% sodium azide, 0.5% BSA (HBB),containing either 2.5 μg THP-1 membrane protein or 500,000 PBMC and 0.1nM of [¹²⁵I]-MCP-1. Competition binding experiments were performed byincluding variable concentrations of unlabeled MCP-1, 1D9 antibody, or anegative control IgG2a. Nonspecific binding was determined following theaddition of a 2500-fold excess of unlabeled MCP-1. Samples wereincubated for 60 minutes at room temperature, and bound and free tracerwere separated by filtration through 96-well GF/B filterplates presoakedin 0.3% polyethyleneimine. The filters were washed in HBB furthersupplemented with 0.5 M NaCl, dried, and the amount of boundradioactivity determined by liquid scintillation counting. mAb 1D9inhibits [¹²⁵I]MCP-1 binding to THP-1 cell membranes with an IC₅₀ ofabout 0.004 μg/ml (approximately 0.02 nM; FIG. 4) and to fresh PBMC withan IC₅₀ of 0.04 μg/ml (approximately 0.2 nM; FIG. 5).

Chemotaxis of PBMC

Chemotaxis was assayed using a 3 μm pore size 96-well chemotaxis plate(Neuroprobe, Cabin John, MD). PBMC isolated by standard methods usingFicoll-Hypaque density gradient centrifugation were washed with PBS/0.5%BSA and then resuspended in chemotaxis assay media (HBSS/10 mMHEPES/0.5% Fatty acid free BSA) to a final concentration of 10×10⁶cells/ml. Cells were princubated in chemotaxis assay media at roomtemperature for 20 min. with various concentrations of the anti-CCR2antibody, 1D9, or nonspecific murine IgG2a. The same dilutions ofantibody were mixed with chemokine and 30 μl of the mixture was added toeach of the bottom wells of the chemotaxis plate. The bottom wells arecovered with the membrane, and 25 μl of the cell and antibody mixtureare added to the top of the filter. The plates are incubated at 37° C.in 5% CO₂ incubator for approximately 80 min. At the completion of themigration, the membrane is removed and the plate with the bottom wellsis incubated −80 C for 30 minutes to freeze the contents. The plates arethawed at 37° C. for 10 minutes. 6 μl of a 1:400 dilution of CyQuantreagent (Molecular Probes, Eugene, Oreg.) in a lysis buffer provided bythe supplier is added to each well, and the cell migration is quantifiedas indicated by fluorescence intensity determined using a CytoFlourfluorescence plate reader at 485ex/535em. mAb 1D9 inhibits MCP-1-inducedchemotaxis, but not RANTES-induced chemotaxis, of fresh PBMC (FIGS. 6Aand 6B). Inhibition of MCP-1-induced chemotaxis of fresh PBMC has beendemonstrated with 10 μg/ml (≈40 nM).

Example 2 Humanisation of Monoclonal Antibody 1D9

The 1D9 monoclonal antibody is likely to be immunogenic in humans,potentially eliciting a human anti-mouse antibody (HAMA) response. ThisHAMA response usually results in rapid clearance of the mouse monoclonalantibody from the body, thus limiting any therapeutic effect the 1D9monoclonal antibody might have. Therefore, in an effort to reduce theimmunogenicity of this antibody in humans and to maximize itstherapeutic potential, the humanisation of the 1D9 mouse monoclonalantibody was undertaken. The following examples provide a detailedanalysis of the 1D9 amino acid sequence data, the building of amolecular model of the murine 1D9 F_(V) domain, and the design strategyfor the successful humanization of the mouse antibody. This designstrategy resulted in the design of a number of humanized versions ofboth the kappa light chain variable (V_(K)) region and the heavy chainvariable (V_(H)) region. In total, the humanized V_(H) region includedup to 16 amino acid changes in the FRs of the selected human V_(H)region. These changes were subdivided between four versions of thehumanized V_(H) region. In addition, twelve amino acid changes in theFRs of the selected human V_(K) region were included in the fourversions of the humanized VK region which were also designed.

Sequence Analysis of the Mouse 1D9 Kappa Light Chain Variable Region

The amino acid sequence of the 1D9 V_(K) region (FIG. 7) was comparedwith other mouse kappa light chain variable regions and also theconsensus sequences of the subgroups that the variable regions weresubdivided into in the Kabat database (Kabat et al., Sequences ofproteins of immunological interest, Fifth edition, U.S. Department ofHealth and Human Services, U.S. Government Printing Office (1991)). Fromthis analysis the 1D9 V_(K) region was found to most closely match themouse consensus sequence of mouse kappa subgroup II (Identity=79.46%,Similarity=82.14%). When only the FRs of the 1D9 kappa light chainvariable region were compared in the mouse subgroup II, percentageidentity increased to 87.5%, while percentage similarity increased to88.75%. In addition, the mouse 1D9 V_(K) region showed good homology toa translation of the 70/3 murine V_(K) germline gene (FIG. 13). Takentogether, the above evidence clearly proved that the 1D9 sequence wastypical of a mouse V_(K) region.

Sequence Analysis of the Mouse 1D9 Heavy Chain Variable Region

A similar analysis of the 1D9 V_(H) region (FIG. 8) found that itmatched closest to the consensus sequence of the mouse heavy chainsubgroup IIIc in the Kabat database (Kabat et al., Sequences of proteinsof immunological interest, Fifth edition, U.S. Department of Health andHuman Services, U.S. Government Printing Office (1991)). Identitybetween the mouse heavy chain variable region amino acid sequence of 1D9and the consensus sequence of mouse subgroup IIIc was measured at70.94%, while the similarity was calculated to be 76.07%, When only theFRs of the 1D9 V_(H) region was compared to mouse subgroup IIIc,percentage identity increased to 75.86%, while the similarity increasedto 80.46%. The mouse 1D9 V_(H) region also showed good homology to atranslation of the MLR-RF24BG murine V_(H) germline gene, among others(FIG. 14). Thus, the above evidence confirmed that the 1D9 sequence wastypical of a mouse V_(H) region.

Molecular Modelling of the 1D9 Domain.

To assist in the design of the humanized variable regions of the 1D9antibody, a series of molecular models were constructed of the murine1D9 F_(V) region and the eight CDR grafted variants. This was done usingthe AbM molecular modeling package supplied and utilized by OxfordMolecular Limited (OML). Antibody x-ray crystallographic structuresavailable from the Brookhaven database were formatted to allow them tobe used for modeling with AbM.

The FRs of the 1D9 variable regions were modeled on FRs from similar,structurally-solved immunoglobulin variable regions. While identicalamino acid side chains were kept in their original orientation,mismatched side chains were substituted as in the original 1D9 F_(V).The backbone atoms of the FRs of the Fab Bv04-01 V_(K) region were usedfor the model of the F_(V) framework region of 1D9 for both the V_(K)and V_(H) chains (Brookhaven PDB code 1nbv, solved to 2.0 Å). Thesequence of Fab Bv04-01 was a good match for the variable regionsequences of murine 1D9 and their humanized variants. The identitiesbetween Fab Bv04-01 and the murine 1D9 and humanized sequences rangedfrom 76% to 78% for V_(K) sequences and from 74% to 84% for V_(H)sequences. Testing of AbM with known structures has shown that FRbackbone homology is an important factor in the quality of any model,since the use of FR structures that poorly match a sequence beingmodeled can significantly and adversely affect the position andorientation of the CDR loops.

For the backbone structures of CDRs L1, L2, L3, H1 and H2, conformationsfor all of the models were taken from canonical classes used by AbMwithout modification, using the classes shown in FIGS. 9 and 10.

For the backbone structure of the L1 loop, the loop conformations of themurine 1D9 V_(K) region was taken from canonical Class 4 from AbM. Thiscanonical class is based on those described by Chothia and hiscolleagues (Chothia and Lesk, J. Mol. Biol. 197:901 (1987); Chothia etal., Nature 34:877 (1989); Tramontano et al., J. Mol. Biol. 215:175(1990); and Chothia et al., J. Mol. Biol. 227:799 (1992)), but they havebeen modified to take into consideration structures that have becomeavailable since the original articles were published. Testing of theperformance of AbM predictions for known loop structures has shown thatCDR loops which are created in this way are usually modeled veryaccurately, i.e. to within 1-1.5 Å RMS deviation.

The H3 loop in the 1D9 V_(H) region is comparatively short at sixresidues long. It was modeled using a search for backbone conformationsfrom X-ray structures in the Brookhaven databank. For short loops likethis, there are sufficient loop conformations from known X-raystructures to saturate the conformational space available to the loop.Testing of the predictions made by AbM with the structures of newantibodies, where the structure is not included in the databases used bythe program, shows that for CDR H3 loops of this size, the accuracy islikely to be at least 2.0 Å.

After adjusting the whole of the model for obvious steric clashes it wassubjected to energy minimisation, as implemented in MACROMODEL, both torelieve unfavorable atomic contacts and to optimize van der Waals andelectrostatic interactions.

Design of the Humanised 1D9 V_(K) Antibody Variants.

The first step in the design of the humanised variable regions of the1D9 antibody was the selection of the human kappa light chain variableregion that would serve as the basis for the humanized 1D9 V_(K),region. As an aid to this process the 1D9 V_(K) region was initiallycompared to the consensus sequence of the four human kappa light chainvariable region subgroups as defined by Kabat (Kabat et al., Sequencesof proteins of immunological interest, Fifth edition, U.S. Department ofHealth and Human Services, U.S. Government Printing Office (1991)). Themouse 1D9 light chain variable region was most similar to the consensussequence of human kappa light chain subgroup II, with which it displayeda 76.2% identity over the whole variable region and a 82.5% identitywithin the FRs alone. When measured with respect to similarity, thesevalues increased to 79.7% overall and 85.0% within the FRs alone.Consequently it generally appeared to match well to human kappa lightchain variable region sequences from kappa subgroup II.

The mouse 1D9 V_(K) was then compared to all the recorded examples ofindividual sequences of human variable regions publicly available. FIG.15 shows the best seventeen matches to the mouse 1D9 V_(K) region whichwere identified through this analysis. Overall, the search algorithmselected the human V_(K) region antibody 036521 (Rheinnecker et al.,Journal of Immunology. 157(7):2989-97 (1996)) as the best match to themouse 1D9 V_(K) region (FIG. 16). However, a review of the source paperfor this antibody revealed that murine oligonucleotide primers had beenused to rescue the genes from the hybridoma. This meant that thisantibody was in fact a murine antibody and not human, as suggested bythe Kabat database. Thus, the next best match to the murine 1D9 V_(K)region that was selected by the database search was the human V_(K)region from antibody HF-21/28 (Kabat database ID number 005056;Chastagner et al., Gene. 101(2):305-6 (1991)). The human sequence had anoverall identity to the 1D9 V_(K) region of 79.3% and 85.0% within theFRs alone. When measured with respect to similarity, these valuesincrease to 83.99% overall and 87.5% within the FRs alone. In addition,key FR amino acids were more conservatively preserved in HF-21/28 V_(K)region than in the other candidate human kappa light chain variableregions. Consequently, the HF-21/28 kappa light chain variable region FRwas selected as the human acceptor sequence for the humanization of the1D9 antibody kappa light chain variable region.

Unfortunately, the very last residue in FR4 (at position 107, accordingto the Kabat numbering system) of the human HF-21/28 V_(K) region wasnot defined by the Kabat database or the authors who originally isolatedthis variable region sequence. Therefore, it was decided to insert themost commonly found amino acid at this position in the variable regionsequences described by Kabat human kappa light chain subgroup κ-II(Kabat et al., Sequences of proteins of immunological interest, Fifthedition, U.S. Department of Health and Human Services, U.S. GovernmentPrinting Office (1991)). Accordingly, lysine was added at position 107in FR4 based upon an analysis of the Kabat database which found that85.7% of the sequences in Kabat human kappa light chain subgroup κ-IIhad a lysine at this position. This then became the basis of the firsthumanized version of the 1D9 kappa light chain(1D9RK_(A)), whichessentially comprised the CDR's of the 1D9 V_(K) region and the FRs ofHF-21/28 V_(K) region. FIGS. 19A-19C define the amino acid sequence ofthis first CDR-grafted version of the humanised 1D9 V_(K) region.

The next step in the design process was to study the amino acidsequences of the human acceptor HF-21/28 V_(K) region FRs to determineif any of these amino acid residues were likely to adversely influencebinding to antigen. This could be caused directly through interactionswith antigen, or indirectly by altering the confirmation or orientationof the CDR loops. This was a difficult process which was only madepossible through the availability of a model of the 1D9 variableregions, i.e., both the V_(K) and the V_(H) regions. Nevertheless, anyamino acids in the mouse 1D9 FRs that did appear to affect antigenbinding were then considered for conversion in the humanized 1D9antibody. In deciding which murine residues to conserve the followingpoints were addressed:

-   -   It was of great importance that the canonical structures of the        hypervariable loops (Chothia and Lesk, J. Mol. Biol. 197:901        (1987); Chothia et al., Nature 34:877-(1989); Tramontano et        al., J. Mol. Biol. 215:175 (1990); and Chothia et al., J. Mol.        Biol. 227:799 (1992)) were conserved. Consequently, it was        crucial to conserve in the humanized 1D9 variable regions all        the mouse FR residues that were part of these canonical        structures (FIGS. 9 and 10).    -   The sequences of the 1D9 antibody variable regions were compared        to similar sequences from other mouse antibodies to identify        unusual or rare residues which may have indicated an important        role in antigen binding. This was then investigated using the        mouse model of the 1D9 variable region genes.    -   A direct analysis of the model was also made to try and predict        whether any of the other mouse FR residues not present in the        humanized FRs could influence antigen binding in some way.    -   Comparisons of the individual human acceptor sequences for the        kappa light chain and heavy chain variable regions to the        consensus sequence of human variable regions subgroups to which        the acceptor sequences belonged were also made. The        identification of any idiosyncratic amino acids in the human        donor sequences was important, as these could have adversely        affected antigen binding.    -   Since the human light and heavy chain variable regions selected        would be derived from two different human antibodies, a careful        analysis of the interdomain packing residues of both the donor        and the acceptor kappa light chain variable regions should be        carried out (Chothia et al., J. Mol. Biol. 186:651 (1985)). This        was because any mispacking in this region could have had a        dramatic affect upon antigen binding, irrespective of the        conformation of the CDR loop structures of the humanized 1D9        antibody.

Although there were 12 amino acid differences between the FRs of thedonor mouse 1D9 V_(K) region and the acceptor human HF-21/28 V_(K)region, only two mouse residues were considered sufficiently importantfor binding affinity to preserve them in the humanised FRs. The first ofthe FR changes that were introduced into 1D9RK_(B) was located atposition 36. This residue is a Vernier residue (Foote and Winter, J.Mol. Biol. 224:487 (1992)) and is predicted to be a key structuredetermining residue for L1 loop structure as defined by Chothia and hiscoworkers (Chothia and Lesk, J. Mol. Biol. 197:901 (1987); Chothia etal., Nature 34:877 (1989); Tramontano et al., J. Mol. Biol. 215:175(1990); and Chothia et al., J. Mol. Biol. 227:799 (1992)). Although bothresidues are hydrophobic, the human Phe is bulkier at this position, andX-ray structures with Leu and Phe at this position show that if Phe ispresent steric hindrance causes the side chain at 34Asn to point in theopposite direction. Thus, it was considered critical for the successfulhumanization of the 1D9 kappa light chain.

The second change incorporated into the 1D9RK_(B) humanized version wasat residue 37, i.e., Gln37Leu. Although this was a conservative changeit occurred in a highly conserved region at the base of CDR1. It wasthought that by preserving this murine Leu residue in this version,alongside the murine Leu residue at position 36, the affinity of thehumanised antibody could be preserved.

Two other versions of the humanised V_(K) region were also consideredfor construction to explore the structural and binding affinityconsequences of manipulating the FRs of the humanised 1D9 antibody.1D9RK_(C) was essentially identical to 1D9RK_(B), except for themutation Gln100Gly. There is a dramatic difference in molecule bulkbetween these two residues, and thus this version was made to explorethe consequences of this change to the FRs of the reshaped human kappalight chain on antibody structure and overall antibody affinity.1D9RK_(D) contained the modifications described in 1D9RK_(C) and, inaddition, contained the FR change Gln17His. Although Gln and His aresimilar in size and both are weakly polar, the mouse residue (His) atthis position is extremely rare amongst all mouse V_(K) sequences (0.07%overall, but has not been seen in mouse Kabat subgroup II sequences) andhas never been seen in any human V_(K) sequences. Conversely, the Glnresidue is more commonly seen at this position in both mouse (16.16%overall and 6.12% in mouse Kabat subgroup II sequences) and human (5.00%overall and 39.7% in human Kabat subgroup II sequences) sequences. Thus,the simple rarity of the His at this position suggests that it may beimportant for binding, although there is no clear evidence to supportthis from the molecular modeling data. A description of the amino acidsequences of all the humanised 1D9 antibody V_(K) region variantsproposed above are given in FIG. 11.

Design of Humanised 1D9 V_(H) Antibody Variants

Once again, the first step in the design of the humanised V_(H) regionof the mouse 1D9 antibody was the selection of the acceptor human heavychain variable region that would serve as the basis of the humanised 1D9V_(H) region. When the 1D9 V_(H) region was initially compared to theconsensus sequences of the three human heavy chain variable regionsubgroups it was found to be most similar to the consensus sequence forthe human heavy chain subgroup III, with a 69.231% identity overall anda 78.161% identity between the FRs alone. When measured with respect tosimilarity, these values increased to 74.359% overall and to 82.759%within the FRs alone.

The mouse 1D9 V_(H) region was then compared to all the recordedexamples of individual sequences of human variable regions publiclyavailable. FIGS. 17A-B show the best 24 matches to the mouse 1D9 V_(H)region which were identified through this analysis. Overall the searchalgorithm selected the human V_(H) region from antibody 4B4′CL (Kabatdata base ID number 000490; Sanz et al., Journal of Immunology. 142:883(1989)) as the best match to the mouse 1D9 V_(H) region. The V_(H)region of this clone had an overall identity to the 1D9 V_(H) region of67.2%, a value which increased to 80.95% when the FRs alone werecompared (FIGS. 18A-B). When measured with respect to similarity, thesevalues increased to 69.66% overall and to 84.52% within the FRs alone.Thus, although once again not the most homologous of the potential humanacceptor V_(H) sequences, this human FR became the basis of thehumanised version of the 1D9 heavy chain.

The next step in the design process was to study the amino acidsequences of the human acceptor 4B4′CL V_(H) region FRs to determine ifany of these amino acid residues were likely to adversely affect bindingto antigen. Once again the molecular models built by OML were importantin this design process, from which a number of amino acids in the murine1D9 V_(H) region FRs were identified for conversion in the first(1D9RH_(A)) and subsequent versions of the humanised 1D9 antibody (FIG.12 and FIGS. 20A-C). There were 16 amino acid differences between theFRs of the donor mouse 1D9 and the acceptor human 4B4′CL V_(H) regions,and up to 5 murine residues were considered for conservation in thehumanised FRs (FIG. 12).

1D9RH_(A) consisted of the CDRs of the murine 1D9 antibody V_(H) regiongenetically inserted into the FRs of the human 4B4′CL antibody V_(H)region. 1D9RH_(B) was identical to version 1D9RH_(A) apart from two FR1mutations, Thr28Ser and Asn30Ser. These changes were made because theyrepresented Vernier amino acids as defined by Foote and Winter (J. Mol.Biol. 224:487 (1992)), which were thought to be critical for H1 loopconformation. Residues 27-30 are considered part of the H1 loop itselfand so are even more critical to the correct conformation andorientation of this loop, justifying their conservation even morestrongly. Thus, these two residues represented the sum of the changesmade to the FRs of the human 4B4′CL V_(H) sequence in 1D9RH_(B).1D9RH_(C) was identical to version 1D9RH_(B) except that it containedtwo further changes at positions Gly49Ala and Phe67Tyr. The Gly49Ala wasa conservative change. However, residue position 49 has been identifiedas a Vernier residue (Foote and Winter, J. Mol. Biol. 224:487 (1992)),important for H2 hypervariable loop structure, so it was decided toconserve the murine Ala residue in this version. Residue position 67 wasalso a Vernier residue position, identifying it as important formaintaining CDR loop conformation. Tyr is very rarely seen in humanV_(H) sequences (0.08% overall) and has not previously been found inmurine V_(H) regions at this position. Consequently, it must have arisenthrough somatic mutation. Thus, given its location close to CDR2according to the molecular model and its Vernier residue status, it wasdecided to conserve the murine Tyr residue at this position. 1D9RH_(D)was identical to 1D9RH_(C) except for a Thr93Val mutation. This residuehad been identified as important as both a V_(H)/V_(K) packing residue(Chothia et al., J. Mol. Biol. 186:651 (1985)). Moreover, its buriedposition between CDR loops H1 and H3, according to the molecular model,supported the decision to conserve the murine Val residue at thisposition. A description of the amino acid sequences of all the humanisedV_(H) region variants described above are given in FIG. 12.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1.-38. (canceled)
 39. A method of inhibiting a function associated withbinding of a chemokine to a mammalian CC-chemokine receptor 2 (CCR2),comprising contacting a composition comprising the CCR2 with aneffective amount of a humanized antibody or antigen-binding fragmentthereof, wherein the antibody or antigen-binding fragment thereofinhibits binding of the chemokine to CCR2 and inhibits one or morefunctions associated with binding of the chemokine to CCR2.
 40. Themethod of claim 39, wherein the chemokine is selected from the groupconsisting of MCP-1, MCP-2, MCP-3, MCP-4 and combinations thereof. 41.The method of claim 40, wherein said function is selected from the groupconsisting of: (a) signaling activity; (b) stimulation of a cellularresponse; and (c) combinations of (a) and (b).
 42. The method of claim41, wherein said function is signaling activity and is selected from thegroup consisting of: (a) activation of a mammalian G protein; (b)induction of a rapid and transient increase in the concentration ofcytosolic free calcium [Ca²⁺]I; and (c) combinations of (a) and (b). 43.The method of claim 41, wherein said function is stimulation of acellular response and is selected from the group consisting of: (a)stimulation of chemotaxis; (b) exocytosis; (c) inflammatory mediatorrelease by leukocytes; (d) integrin activation; (e) T cell activation;(f) leukocyte degranulation; and (g) combinations of (a), (b), (c), (d),(e) and (f).
 44. The method of claim 39, wherein the humanized antibodyor antigen-binding fragment thereof comprises three complementaritydetermining region sequences of the light chain of the 1D9 antibody anda framework region sequence of the variable light chain of the HF21/28antibody.
 45. The method of claim 39, wherein the humanized antibody orantigen-binding fragment thereof comprises three complementaritydetermining region sequence of the variable heavy chain of monoclonalantibody 1D9 and a framework region sequence of the variable heavy chainof the 4B4′CL antibody.
 46. The method of claim 44, wherein thehumanized antibody or antigen-binding fragment thereof comprises threecomplementarity determining region sequences of the variable heavy chainof monoclonal antibody 1D9.
 47. The method of claim 46, wherein thehumanized antibody or antigen-binding fragment thereof further comprisesa framework region derived from the variable heavy chain of the 4B4′CLantibody.
 48. The method of claim 39, wherein the humanized antibody orantigen-binding fragment thereof comprises three complementaritydetermining region sequence of the variable light chain of monoclonalantibody 1D9, a framework region sequence of the variable light chain ofthe HF 21/28 antibody, three complementarity determining region sequenceof the variable heavy chain of monoclonal antibody 1D9 and a frameworkregion sequence of the variable heavy chain of the 4B4′CL antibody. 49.The method of claim 48, wherein the humanized antibody orantigen-binding fragment thereof comprises a heavy chain constant regionor portion thereof.
 50. The method of claim 49, wherein the humanconstant region or portion thereof is of the gamma type.
 51. The methodof claim 50, wherein the human constant region or portion thereof ismutated to minimize binding to Fc receptors, the ability to fixcomplement or both.
 52. The method of claim 48, wherein the humanizedantibody or antigen-binding fragment thereof, comprises a light chainconstant region.
 53. The method of claim 52, wherein the human lightchain constant region is of the kappa type.
 54. The method of claim 48,wherein the light chain variable region of the humanized antibody orantigen-binding fragment thereof comprises the amino acid sequence ofSEQ ID NO:12.
 55. The method of claim 48, wherein the heavy chainvariable region of the humanized antibody or antigen-binding fragmentthereof comprises the amino acid sequence of SEQ ID NO:17.
 56. Themethod of claim 48, wherein the light chain variable region of thehumanized antibody or antigen-binding fragment thereof comprises theamino acid sequence of SEQ ID NO: 12, and the heavy chain variableregion of the humanized antibody or antigen-binding fragment thereofcomprises the amino acid sequence of SEQ ID NO:17.
 57. The method ofclaim 56, wherein the humanized antibody or antigen-binding fragmentthereof, comprises a heavy chain constant region or portion thereof. 58.The method of claim 57, wherein the human constant region or portionthereof is of the gamma type.
 59. The method of claim 58, wherein thehuman constant region or portion thereof is mutated to minimize bindingto Fc receptors, the ability to fix complement or both.
 60. The methodof claim 56, wherein the humanized antibody or antigen-binding fragmentthereof, comprises a light chain constant region.
 61. The method ofclaim 60, wherein the human light chain constant region is of the kappatype.